1
Introduction
Nitrogen is an essential
element contained in many biomolecules
necessary to sustain life.
1,2
This element is abundantly
available in Earth’s atmosphere in the form of dinitrogen (N2) gas, yet most organisms
are unable to metabolize N2 because it is relatively inert.
3,4
Instead most
organisms must obtain their N from “fixed” forms such
as ammonia (NH3) or nitrate (NO3
–).
5−7
Because fixed forms of N are continuously sequestered into sediments,
rendering them unavailable for metabolism, and because they are also
continuously converted to N2 through the combined processes
of nitrification and denitrification, life can only be sustained by
conversion of N2 to NH3.
6,7
This
latter process is known as N2 fixation
8
and is a critical step in the biogeochemical N cycle.
5,7,9
N2 fixation occurs
in three different ways: (i) through geochemical processes such as
lightning,
9
(ii) biologically through the
action of the enzyme, nitrogenase,
10,11
found only
in a select group of microorganisms,
12,13
and (iii)
industrially through the Haber–Bosch process.
2,14,15
From the evolution of nitrogenase,
approximately two billion years ago
16
until
the widespread use of the Haber–Bosch process in the 1950s,
all life derived N from biological nitrogen fixation, with geochemical
processes representing a minor contributor to the supply of fixed
nitrogen.
2,7
Since the increase in use of the Haber–Bosch
process, the biological and industrial processes contribute comparably
to N2 fixation.
5,7,9
Nitrogen fixation has a profound agronomic, economic, and
ecological
impact owing to the fact that the availability of fixed nitrogen represents
the factor that most frequently limits agricultural production throughout
the world.
2
Indeed, nearly half of the
existing human population could not exist without application of the
Haber–Bosch process for production of nitrogen fertilizers.
2,5
Given that over half of the fixed nitrogen input that sustains Earth’s
population is supplied biologically, there has been intense interest
in understanding how the nitrogenase enzyme accomplishes the difficult
task of N2 fixation at ambient temperature and pressure.
17,18
An understanding of biological N2 fixation may further
serve as the foundation for achieving two highly desirable, although
so far unmet, goals: genetically endowing higher plants with the capacity
to fix their own nitrogen,
19−21
and developing improved synthetic
catalysts based on the biological mechanism.
3,4,22−25
It has been over 150 years
since Jodin first suggested that microbes
could “fix” N2,
26
and more than a century since the first isolation of N2-fixing bacteria around 1900.
In 1934, Burk coined the term “nitrogenase”
10,11
for the enzyme that catalyzes the conversion of N2 to
a bioaccessible form of nitrogen, and initiated the first meaningful
studies of nitrogenase in living cells. Methods for extracting nitrogenase
in an active form were developed in the early 1960s,
27−29
opening the way for serious mechanistic investigations. The next
35 years witnessed intensive efforts by numerous investigators to
reveal the structure and catalytic function of nitrogenase.
30−34
These developments were summarized in the magisterial review by
Burgess and Lowe in 1996.
17
Key advances
in understanding nitrogenase structure and function during those intervening
years included the following: (i) It was determined that nitrogenase
is a two-component system
35−37
composed of the MoFe protein
(also called dinitrogenase or component I) and the electron-transfer
Fe protein (also called dinitrogenase reductase or component II).
34,38−41
(ii) A reducing source and MgATP are required for catalysis.
42−45
(iii) Fe protein and MoFe protein associate and dissociate in a
catalytic cycle involving single electron transfer and MgATP hydrolysis.
38
(iv) It was discovered that the MoFe protein
contains two metal clusters: the iron–molybdenum cofactor (FeMo-co),
30,46
which provides the active site for substrate binding and reduction,
and P-cluster, involved in electron transfer from the Fe protein to
FeMo-co.
39,47−50
(v) Crystallographic structures
were solved for both Fe
51
and MoFe
32,48,52−54
proteins. (vi)
Also, the alternative V- and Fe-type nitrogenases, in which the Mo
of FeMo-co is replaced by V or Fe, were discovered.
18
Despite this accumulation of functional and structural
information, the catalytic mechanism remained elusive.
The years
since the Burgess and Lowe review
17
have
seen profound advances in understanding many aspects
of nitrogenase structure and function. For example, the solutions
of a number of high-resolution X-ray structures of the nitrogenase
component proteins
55−69
have provided insights into the nature of the active site FeMo-cofactor,
most recently identifying the presence of an interstitial C atom,
70−77
while structures of the two proteins in the complex
78−81
have identified their binding interface (Figures 1 and 2) and its alterations with
the
state of the bound nucleotide.
67
Likewise,
great strides have been made in understanding the biosynthesis and
insertion of the metal clusters of nitrogenase to form the mature
proteins,
21,82−89
and the properties of the V-type nitrogenase.
90−98
Recent studies have begun to shed light on the order of events during
the catalytic cycle,
99−103
including the nature of electron transfer between the metal clusters
62,104−111
and the roles of ATP binding and hydrolysis in these processes.
55,68,99,112−121
Considerable progress has been made in the application of theoretical
methods to various aspects of the nitrogenase mechanism.
122−140
Finally, progress has been made in expanding in the substrates of
nitrogenases
93,141−149
to include CO
95,96,98,150,151
and CO2.
148,152
Figure 1
Molybdenum nitrogenase. (A) One catalytic
half of the Fe protein:MoFe
protein complex with the Fe protein homodimer shown in tan, the MoFe
protein α subunit in green, and the β subunit in cyan.
(B) Space filling and stick models for the 4Fe–4S cluster (F),
P-cluster (P), and FeMo-co (M). Made with Pymol and ChemDraw using
PDB:2AFK.
Figure 2
FeMo-cofactor and the side chains of selected amino acid residues
of the MoFe protein. Numbering of iron atoms is according to the structure
PDB coordinate 2AFK. Iron is shown in rust, molybdenum in magenta, nitrogen in blue,
sulfur in yellow, carbon in gray, and oxygen in red.
The present narrative focuses on recent progress
in understanding
the mechanism of N2 activation and reduction to ammonia
by Mo-nitrogenase. The discussion begins with a short reminder of
the kinetic scheme that describes nitrogenase catalysis.
33,103
It then turns to the successes in trapping catalytic intermediates
of the MoFe protein by rapid freezing of turnover mixtures of Fe protein
and of MoFe proteins, both wild-type and variants containing selected
amino acid substitutions as a means to modulating reactivity.
146,148,153,154
The use of EPR/ENDOR/ESEEM spectroscopic techniques applied to isotopically
substituted trapped intermediates has allowed the identification and
characterization of key intermediates along the N2 reduction
pathway.
154−156
This led to the formulation of a reaction
mechanism based on the properties of catalytic intermediates and grounded
in the reaction of hydrides associated with FeMo-co.
156
The mechanism not only satisfies all constraints on the
mechanism provided by earlier studies, but has suggested and
passed a stringent test.
157
This
report recounts these advances, and expands on them.
2
Background
Two issues require consideration as a basis
for discussion of recent
advances in nitrogenase mechanism.
155,156
The first
is the kinetic model that has been developed to describe the multistep
reduction of N2 to two NH3, and its implications
for the stoichiometry of this reaction,
33,103
implications
that were mutually supported by experiment.
158
The second is the strategies and procedure that at last enabled
the trapping of catalytic intermediates whose characterization by
advanced paramagnetic resonance techniques underlies the progress
in mechanism described here.
2.1
Kinetics and Stoichiometry
A “kinetic”
foundation for a nitrogenase mechanism was developed by extensive
studies in the 1970s and 1980s by many groups, especially Lowe and
Thorneley and their co-workers.
17,33,103
The culmination of these extensive kinetic studies, which involved
steady-state, stopped-flow, and freeze–quench kinetics measurements,
was the Lowe–Thorneley (LT) kinetic model for nitrogenase function,
17,33,103
which describes the kinetics
of transformations among catalytic intermediates (denoted E
n
) where n is the number of
steps of electrons/protons delivery to MoFe protein, Figure 3. Electron transfer from
Fe protein to MoFe protein
is driven by the binding and hydrolysis of two MgATP species within
the Fe protein;
99
the release of the Fe protein after delivery of its electron is the rate-limiting
step of catalysis.
33
Figure 3
Simplified LT kinetic
scheme that highlights correlated electron/proton
delivery in eight steps. Although in the full LT scheme N2 binds at either the E3
or E4 levels, the pathway
through E3 is de-emphasized here. LT also denotes the protons
bound to FeMo-co (e.g., E1H1); for clarity we
have omitted these protons in this scheme.
A central consequence of the kinetic measurements and defining
feature of this scheme, Figure 3, is that the
limiting enzymatic stoichiometry for enzyme-catalyzed nitrogen fixation
is not what would be given by the simple balanced equation for reduction
of N2 to two NH3 by six electrons/protons, but
is given by eq 1
1
This is a conclusion that is in agreement
with stoichiometric experiments by Simpson and Burris.
158
This equation highlights several key aspects
of the nitrogenase mechanism, including the involvement of ATP hydrolysis
in substrate reduction and the obligatory formation of 1 mol of H2 per mole of N2
reduced, an apparent “waste”
of two reducing equivalents and four ATP per N2 reduced.
17,33
Although the close of the previous millennium saw the accumulation
of a vast breadth and depth of information about the reduction of
N2, H+, and a variety of other nonphysiological
substrates,
17
it was not until recently
that studies have succeeded in characterizing E
n
intermediate states beyond the resting-state E0.
154−156
Thus, the early studies provided little
direct experimental evidence regarding a reaction pathway, and hence,
there was no possibility of integrating a reaction pathway and kinetic
scheme, as is central to development of a mechanism based on the properties
of catalytic intermediates.
156
2.2
Trapping and Characterization of Substrates
The first
40 years of study of purified nitrogenase did not see
the definitive characterization of any intermediates associated with
the binding and reduction of N2,
159
leaving the identity of the reaction pathway unresolved. The way
forward was provided by studies of nitrogenases with individual amino
acid substitutions, which revealed that the residue at position α-70
within the MoFe protein, a valine, acts as a “gatekeeper”
that sterically controls the access of substrate to the active site
FeMo-co (Figure 2).
146,154
The side chain of this amino acid residue is located over one FeS
face of FeMo-cofactor (that includes Fe atoms 2, 3, 6, and 7) thereby
also implicating Fe as the site of substrate binding, while the α-195His was inferred
to be involved in proton delivery (Figure 2).
160−165
Use of MoFe protein substituted at one or both of these residues
enabled freeze–quench trapping of a number of nitrogenase turnover
intermediates, almost all of which show an EPR signal arising from
an S = 1/2 state of FeMo-co,
rather than the S = 3/2 state
of resting-state FeMo-co.
153,154
The procedures developed
with these variants even enabled N2−intermediate
trapping with enzyme.
166
The first fruit
of this approach was the trapping of a state during reduction of the
alkyne propargyl alcohol to the corresponding alkene.
145,167
An intermediate trapped using MoFe protein variants was shown by
ENDOR studies to be a wholly novel bio-organometallic structure in
which the alkene product of alkyne reduction by nitrogenase binds
as a π-complex/ferracycle to a single Fe ion of FeMo-cofactor,
presumed to be Fe6.
168
This was followed
by characterization of intermediates formed during the reduction of
H+ under Ar,
169
and, finally,
identification of four associated with N2 reduction itself.
147,153,160,166,170,171
Paramagnetic resonance methods have proven to be uniquely
advantageous
for characterization of trapped nitrogenase intermediates.
155
At the most basic level, FeMo-co in the E0 resting-state of MoFe protein is an odd-electron
(“Kramers”;
half-integer spin, S = 3/2
172
), EPR-active cluster, and therefore, intermediate
states that have accumulated an even number of electrons also will
be EPR-active. Focusing on nitrogen fixation, FeMo-co then will be
EPR-active in the E
n
states, n = 2, 4, 6, 8, formed along the pathway for accumulation of the stoichiometrically
required eight [e–/H+], eq 1. In contrast, E
n
states n = 1, 3, 5, 7 will be even-electron, and FeMo-co will either
be diamagnetic or in an integer-spin (“non-Kramers”
spin-state)
173
cluster, which also can
be EPR-active under appropriate conditions.
173,174
As will be illustrated below, electron–nuclear double-resonance
(ENDOR) spectroscopy,
175,176
supported by related techniques
ESEEM and HYSCORE,
177
is uniquely suited
for the study of freeze–quench trapped intermediates. These
techniques give NMR-like spectra of nuclei that are hyperfine-coupled
to the electron spin of an EPR-active cluster. The importance of the
techniques rests on several aspects. ENDOR is broad-banded: with isotopic enrichment
it can monitor every atom
in a metalloenzyme active site. Thus, when interpreted in the context
of the X-ray structure of the resting-state, it can reveal the electronic
and metrical structure of a catalytic intermediate. It is selective: it interrogates
only EPR-active states. It is high-resolution: it can resolve and interrogate the
signals
from multiple distinct EPR-active centers. It is sensitive: we have successfully analyzed
the properties of intermediates present
in ∼20% abundance in a sample containing ∼100 μM
MoFe protein. Viewed another way, ENDOR is capable of selecting and
characterizing a small fraction of the MoFe protein in a sample. In
contrast, for example, Mössbauer and X-ray absorption techniques,
which have made enormous contributions to the study of resting-state
nitrogenase, interrogate all FeMo-co in a sample, and if the state
of interest is a small minority, its signal is buried and lost. Recently,
however, an X-ray spectroscopic study has given information about
a freeze–quenched nitrogenase intermediate.
178
3
Intermediates of Nitrogenase
Activation
According to the simplified LT kinetic scheme
of Figure 3, the first four of the eight [e–/H+] of nitrogen fixation accumulate prior
to N2 binding, which occurs at the E4 stage.
The complete scheme
17,33,103
allows for N2 binding at E3 as well, but E4 uniquely places the enzyme on the pathway
to N2 hydrogenation.
3.1
E1–E3
The E1 state contains one-electron reduced
cofactor, and
has been assigned as an integer-spin species on the basis of Mössbauer
studies of MoFe protein trapped during turnover under N2.
179,180
High-spin EPR signals (S = 3/2), denoted as 1b and 1c, thought to be
associated with E
n
states, n ≤ 4, were first observed 35 years ago for samples of wild-type
nitrogenase trapped during turnover using a variety of conditions,
181
and more recently were studied by rapid freeze–quench
EPR.
182
The kinetics of appearance of 1b
and 1c demonstrated that they must be assigned to reduced states of
cofactor, n > 1, rather than just as conformers
of
the FeMo-co resting-state. However, the kinetics of appearance of
the stronger 1b signal was a puzzle: they were best described by assigning
1b to the E3 state, which would seem to require that FeMo-co
be in an integer-spin (non-Kramers) state, contrary to observation.
Most likely, this apparent contradiction reflects uncertainties in
the rate constants used in the kinetics analysis, and 1b represents
an E2 state. During cryoannealing experiments
183
discussed below, we definitively observed that
FeMo-co of E2 is in a high-spin (S = 3/2) state, but at least in the α-70Ile variant
its g-values were distinct from those of
1b. The spectrum of the 1c species is weaker in intensity. It may
represent a conformer of the resting or 1b states, or may correspond
to even more reduced states, as its effective formation requires a
high molar ratio of Fe protein to MoFe protein, corresponding to higher
electron flux.
3.2
E4: The “Janus
Intermediate”
In this subsection we describe the trapping
and EPR/ENDOR characterization
of the E4 intermediate as activated by the accumulation
of four [e–/H+] for binding and reduction
of N2. The structure of E4 as determined by
ENDOR spectroscopy, and integrated into the LT kinetic scheme, has
been the key to recognizing the central role of hydrides in the mechanism
for nitrogen fixation.
156
We then discuss
the E1–E4 states associated with electron
accumulation by MoFe protein; subsequent sections discuss the trapped
states associated with the N2 reduction pathway following
N2 binding.
Early in the search for intermediates,
146
the α-70Val→Ile substitution
in the MoFe protein was shown to deny access of all substrates to
the active site, except protons.
169,184
Samples of
this substituted MoFe protein freeze–quenched during turnover
under Ar exhibited a new S = 1/2 EPR signal,
169
which also can be observed
at lower concentrations during turnover of wild-type MoFe protein
under Ar.
181,185
1,2H ENDOR spectroscopic
analysis of this trapped state
169
revealed
the presence of two strongly hyperfine-coupled, metal-bridging hydrides
[M–H–M′]: (i) The finding that
the bound hydrides have a large isotropic hyperfine coupling, a
iso ≈ 24 MHz, led to their assignment
as hydrides bound to metal ion(s) of the core. (ii) The anisotropic hyperfine contribution,
T = [−13.3,
0.7, 12.7] MHz, exhibits almost complete rhombicity, as defined by
the form T
rh
≈ [t, 0, −t]. This form rules out terminal
hydrides, which would have a roughly axial T,
186
and is precisely the form first predicted
187
and then confirmed
188
to be associated with a hydride bridging two paramagnetic metal
ions, namely as [Fe–H–Fe] and/or [Mo–H–Fe]
fragments.
95Mo ENDOR measurements subsequently established
that
both hydrides bridge two Fe ions, forming two [Fe–H–Fe]
fragments (Figure 4), as follows.
189
Equations for the anisotropic hyperfine interaction
matrix, T, of a nucleus that undergoes through-space
dipolar interactions to two spin-coupled metal ions
187
were generalized to describe an arbitrary [M1–H–M2] fragment of a spin-coupled cluster.
The components of T are a function of the [M1–H–M2] geometry and of the coefficients
[K
1, K
2] that
describe the projection of the total cluster spin on the two local
M-ion spins. The 95Mo ENDOR measurements of the intermediate
showed a very small isotropic hyperfine coupling, a
iso(95Mo) ∼ 4 MHz, which indicated that K
Mo is too small to yield the rhombic dipolar
coupling, T
rh
, observed in this
intermediate.
189
The model for E4 displayed in Figure 4 is completed by placement
on sulfurs of the two protons
190,191
that form part of
the delivery of 4[e–/H+] (Figure 3). The protons are so placed because they must be
near to the negative charge density associated with the hydrides in
order to obtain the electrostatic stabilization implicit in the required
accumulation of one proton for each electron delivered to MoFe protein;
17
other arrangements are possible, such as putting
both protons on doubly bridging sulfur, but see below.
Figure 4
Depiction of E4 as containing two [Fe–H–Fe]
moieties, emphasizing the essential role of this key “Janus
intermediate”, which comes at the halfway point in the LT scheme,
having accumulated four [e–/H+], and
whose properties have implications for the first and second halves
of the scheme. Janus image adapted from http://www.plotinus.com/janus_copy2.htm. Figure
adapted with permission from ref (156). Copyright 2013 American Chemical Society.
Cryoannealing this “dihydride”
intermediate in the
frozen state at −20 °C, which prevents further delivery
of electrons from the Fe protein, showed that it relaxes to the resting
FeMo-co state by the successive loss of two H2 molecules.
183
According to the LT scheme, only E4 would undergo this two-step relaxation process
(Scheme 1), with the first relaxation step of E4 yielding H2 and the E2 state, the
second step
returning FeMo-co to the E0 stage with loss of a second
H2, and the production of H2 being revealed
by solvent kinetic isotope effects in both stages. This relaxation
protocol thus revealed that the trapped intermediate is the E4 state, which has accumulated
n = 4 electrons
and protons.
183
As the relaxation measurements
involved tracking the kinetically linked conversion of E4 into E2, and the conversion
of E2 into resting-state
E0, the measurements further allowed an unambiguous identification
of the EPR signal associated with E2 (see above).
Scheme 1
Examination of the simplified version of the LT scheme
of Figure 3 reveals that E4 is a
key stage in the
process of N2 reduction.
33,103
Indeed, we
have denoted it as the “Janus” intermediate, referring
to the Roman God of transitions who is represented with two faces,
one looking to the past and one looking to the future (Figure 4).
156
Looking “back”
from E4 to the steps by which it is formed, E4 is the culmination of one-half of the
electron/proton deliveries
during N2 fixation: four of the eight reducing equivalents
are accumulated in E4, before N2 even becomes
involved. Looking “forward”, toward NH3 formation,
E4 is the state at which N2 hydrogenation begins,
and it is involved in one of the biggest puzzles in N2 fixation:
“why” and “how” H2 is lost
upon N2 binding.
To date, we have visualized E4 by placing its two hydrides
on the Fe2, 3, 6, 7 face of resting-state FeMo-co and sharing a common
vertex at Fe6, Figure 4. Although the hydrides
may well exhibit fluxionality at ambient temperature, their ability
to adopt a configuration with a common vertex is required by the reductive elimination
(re) mechanism of reversible H2 release upon N2 binding (section
7), and Fe6 is favored from earlier kinetic studies on MoFe
protein variants.
69,144,145,154,166,168
However, this model is only
one of four possible configurations based on the resting structure
that have two hydrides sharing an Fe6 vertex. To visualize these structures
we have built the bound hydrides onto the crystal structure of resting-state
FeMo-co using Fe–H distances from model complexes,
188,192
Figure 5.
Figure 5
Mockups of the “Janus” E4 intermediate
in which the two bridging hydrides [Fe–H–Fe] revealed
by ENDOR spectroscopy are built onto the resting-state crystal structure.
These models of FeMo-co have Fe6 as a “vertex” for the
two bridging hydrides to facilitate reductive elimination. The figure
was generated using the coordinate file PDB:2AFK. Iron is shown in
rust, molybdenum in magenta, sulfur in yellow, carbon in dark gray,
and hydrogen in light gray.
Quantum chemical computations will test these alternatives.
However,
the experimentally determined relative orientation of the hyperfine
tensors of the two hydrides provides a significant constraint on their
placement within E4. Given the stability of the FeMo-co
structure that is likely imparted by the interstitial carbide, it
seemed plausible to us that consideration of the constructed models
of Figure 5 would allow us to test these alternative
hydride distributions, even though it is beyond doubt that the structure
of FeMo-co will distort upon substrate binding. This exercise (see Supporting Information)
provides support for
the topology of hydride binding pictured for the
Janus E4 intermediate in Figure 4, with hydrides bridging Fe2/Fe6 and Fe6/Fe7 (Figure
5A,B), as opposed to Figure 5C,D, but
does not discriminate between the structures of Figure 5A,B. In discussions below,
we retain the placement of the
E4 hydrides shown in Figure 4 (Figure 5A) as being more readily visualized in discussions
of mechanism.
The characterization of the hyperfine interactions
of the metal-ion
core of E4 that began with the 95Mo ENDOR measurements
189
was completed by an ENDOR study of the 57Fe atoms of the E4 FeMo-co through use of
a suite
of advanced ENDOR methods.
193
The determination
of hyperfine interactions for two ligand hydrides and all eight metal
ions of FeMo-cofactor in this state will provide the experimental
test that guides future computational studies that seek to characterize
the geometric and electronic structure of E4.
Storage
of the reducing equivalents accumulated in the E4 state
as bridging hydrides has major consequences. A bridging hydride
is less susceptible to protonation than a terminal hydride, and thus
bridging hydride(s) diminish the tendency to lose reducing equivalents
through the formation of H2 (Scheme 1), thereby facilitating the accumulation of reducing
equivalents
by FeMo-co. This mode also lowers the ability of the hydrides to undergo
exchange with protons in the environment, a characteristic that is
shown to be of central importance below. However, the bridging mode
also lowers hydride reactivity toward substrate hydrogenation, relative
to that of terminal hydrides.
194,195
As a result, substrate
hydrogenation most probably incorporates the conversion of hydrides
from bridging to terminal binding modes.
196
We next discuss how the structure found for E4 guides
assignment of structures for the E1–E3 states. Subsequently, we show how the E4 structure
defined
possible mechanisms for coupling H2 loss to N2 binding.
3.3
Redox Behavior and Hydride
Chemistry of E1–E3: Why Such a Big Catalytic
Cluster?
Given that the four accumulated electrons of E4 reside
not on the metal ions but, instead, are formally assigned to the hydrides
of the two Fe-bridging hydrides, what then are the proper descriptions
of E1–E3? The addition of one electron/proton
to the MoFe protein results in the E1 state, and a Mössbauer
study of nitrogenase trapped during turnover under N2
180
suggested that this state contains the reduced
metal-ion core of FeMo-co, denoted M– in Figure 6A. The presence in E4 of two bridging
hydrides/two protons led us to propose that upon delivery
of the second electron/proton to form E2 the metal–sulfur
core of the FeMo-cofactor “shuttles” both electrons
onto one proton to form an [Fe–H–Fe] hydride, leaving
the second proton bound to sulfur for electrostatic stabilization
and the core formally at the resting-state, M0, redox level
(also commonly referred to as, MN),
197
Figure 6A. A subsequent, analogous,
two-stage process would then yield the E4 state, with its
two [Fe–H–Fe] hydrides, two sulfur-bound protons, and
the core at the resting-state, M0, redox level.
193
Figure 6
Formulations of E1–E4 derived
from
consideration of E4 as containing two bound hydrides and
two protons. (A) Assuming reduction of the core in n = 1, 3 states. (B) Alternative
formulation of E1–E4 under the assumption of hydride formation at every stage,
in which case the core is formally oxidized for E
n
, n = 1, 3. Symbols: M represents FeMo-co
core; superscripts are charge difference between core and that of
resting-state (commonly denoted MN); the number of bound
protons/hydrides are indicated. Adapted with permission from ref (156). Copyright
2013 American
Chemical Society.
Such a process of acquiring
the four reducing equivalents of E4 involves only a single
redox couple connecting two formal
redox levels of the FeMo-co core of eight metal ions;
M0 the resting-state, and M– the one-electron
reduced state of the core, Figure 6A.
193
Indeed, comparisons of the 57Fe
ENDOR results for the E4 intermediate with earlier 57Fe ENDOR studies and “electron
inventory analyses”
155,198
of nitrogenase intermediates led us to the remarkable suggestion
that, throughout the nitrogenase catalytic cycle, the FeMo-cofactor
would cycle through only two formal redox levels of the metal-ion
core. On reflection, it seems obvious that only by “storing”
the equivalents as hydrides is it possible to accumulate so much reducing
power at the constant potential of the Fe protein. We further proposed
that such “simple” redox behavior of a complex metal
center might apply to other FeS enzymes carrying out multielectron
substrate reductions.
193
Considering
the critical role of hydrides in storing reducing equivalents,
we also suggested that the E1 and E3 states,
respectively, might well contain one and two bridging hydrides bound
to a formally oxidized metal-ion core (Figure 6B),
100
in which case
the single redox couple accessed would formally be that between M0 and M+. In section
9,
below we adopt this “oxidative” formulation of the E1–E3 structures. We emphasize
that a third
formulation of E1(E3), with hydride(s) bound
to M0 and the presence of oxidized P-cluster, is ruled
out by the absence of EPR signals from P+ in samples trapped
under turnover conditions.
If the FeMo-cofactor does not utilize
more than one redox couple
during catalysis, then why is it constructed from so many metal ions?
As discussed above, the hydrides of E4 bind to at least
two, and plausibly three Fe atoms of a 4-Fe face of FeMo-co, as shown
in Figures 4 and 5.
It is further possible that catalysis is modulated by the linkage
of Fe ion(s) to the anionic atom C that is centrally located within
the metal–sulfur core of the FeMo-cofactor.
70,71
Formation of such a 4Fe face and the incorporation of C is not likely
with less than a trigonal prism of six Fe ions linked by sulfides
to generate these structural features. In this view, the trigonal
prismatic FeMo-cofactor core of six Fe ions plus C generates the catalytically
active 4Fe face. This prism is capped, and its properties are likely
“tuned”, by two “anchor” ions, one Fe
plus a Mo, or a V or Fe in the alternative nitrogenases.
Finally,
and far from least, as we have consistently noted (see section 7), there is good reason
to imagine N2 and/or
the N2H
x
reduction
intermediates may interact with multiple Fe ions on a FeMo-co face.
3.4
Why Does Nitrogenase Not React with H2/D2/T2 in the Absence of N2?
The following question is commonly raised: If electrons
accumulated in E
n
intermediates, n = 2–4, can relax to E
n-2 through formation and release of H
2
during
turnover, as captured in the partial LT scheme, Scheme 1, why does the enzyme not
exhibit the reverse of this reaction,
and react with H2/D2 in what might appear to
be the “microscopic reverse” of H2 release?
We have proposed that H2 formation involves protonation
of an [Fe–H–Fe], and at a basic level, all three relaxation
processes of Scheme 1 should have much the
same characteristics. For simplicity in addressing this issue, we
focus on the “first” of these, the E2 →
E0 relaxation, and ask why E0 is not reduced
by H2 to form E2, eq 2
2
A logical answer to this question begins with
the recognition that the LT kinetic scheme for N2 fixation,
Figure 3 (also denoted the “MoFe protein
cycle”), and the segment presented in Scheme 1, omit the reactions of the Fe protein
for clarity; these
are treated as a separate “Fe-protein cycle”.
17,33,103,154
A stoichiometrically correct scheme that merges the Fe protein and
MoFe protein cycles is given in Figure 7. It
reminds us that E2 is formed by two steps of Fe →
MoFe protein ET, with each step involving hydrolysis of two ATP molecules
to drive a reaction that is highly “uphill” energetically.
Figure 7
Formation
and relaxation of E2. In-line: The “on-path”
two-step, ATP-dependent addition of two H+/e– to MoFe protein to form E2. Off-line:
Representation
of the exergonic (free energy, +|ΔG
h|) “off-path” relaxation of E2, liberating
H2 and directly regenerating E0 without intervention
of Fe protein, and of the energetically (free energy, +|ΔG
h|) and kinetically forbidden reverse of this
process; E0′ is a putative intermediate state that
causes the reaction of E0 not to be the microscopic reverse
of the release of H2 from E2 (see text).
Clearly the E2 →
E0 relaxation with
accompanying loss of H2 is not the “reverse”
of the turnover formation of E2 from E0; neither
Fe protein reduction nor ATP formation is involved. Instead, it is
a side-reaction of E2. Indeed, it is even quite unlikely
that a direct reaction of H2 with E0 to form
E2 (eq 2) would be the microscopic
reverse of the E2 → E0 relaxation with
accompanying loss of H2. Moreover, the steric congestion
caused by the sulfurs at the six tetrahedral [FeS3C] sites
of the FeMo-co “waist” requires that the core must relax
for the Fe to bind any ligand; in particular it is probable that the
structure of the [Fe7S9MoC] core of FeMo-cofactor
of E0 (denoted MN) is altered during the reduction
of E0 by two [e–/H+] to form
E2 (also see section 9.2). In
this case, as illustrated in Figure 7, the
relaxation of E2 with loss of H2 to form the
resting E0 state would be a 2-step process. The loss of
H2 by E2 would be expected to form a state (denoted
here E0′) that contains FeMo-cofactor in a conformation
approximating that of E2, corresponding to a metastable
conformation of its resting redox level (denoted MN′); this conformer would in turn
undergo a MN′ →
MN structural relaxation associated with E0′
→ E0 relaxation. The reduction of E0 by
H2 (eq 2) by the microscopic reverse
of this two-step relaxation would correspondingly take place in two
steps, Figure 7, with the initial E0 → E0′ thermal activation associated with
the conformational change, MN → MN′, adding an activation free energy (denoted |ΔG
†|) and kinetic barrier to the endergonic reduction
of E0′ → E2 by H2 (free
energy denoted |ΔG
h′|).
What would be the free energy for reduction of nitrogenase by H2, as in eq 2? An upper
bound for the
free energy change for this reaction, ΔG
h2, would be 4 times the negative of the free energy change
for the hydrolysis of ATP to form ADP and Pi (−ΔG
Hyd ∼ +7 kcal/mol; total, endergonic
by ∼ +28 kcal/mol), that is required for the formation of E2 through the delivery
of reducing equivalents by Fe protein;
roughly compatible with that, oxidative addition of H2 to
an Fe–S center (hydrogenase), corresponding to |ΔG
h′|, is uphill by at least +20 kcal/mol,
199,200
to which must be added the conformational free energy |ΔG
†|. Given the strongly endergonic nature
of eq 2, coupled with the kinetic penalty associated
with the activation of E0 to E0′, it
becomes clear why H2 is not observed to reduce FeMo-cofactor.
4
“Dueling” N2 Reduction
Pathways
Researchers have long considered two competing proposals
for the
second half of the LT kinetic scheme, the reaction pathway for N2 reduction that begins
with the Janus E4 state.
17,139,155
These invoke distinctly different
intermediates, Figure 8, and computations suggest
they likely involve different metal-ion sites on FeMo-co.
139
The “distal” (D) pathway is associated
with the Chatt
4,201
or Chatt–Schrock cycle
3
because it is utilized by inorganic Mo complexes
discovered by these investigators to cleave N2 (Chatt and
co-workers
202,203
) and, most dramatically, to
catalytically fix N2 (Schrock and co-workers
24,204−206
). In this cycle, which has been suggested
to apply to nitrogen fixation by nitrogenase with Mo as the active
site,
139
a single N of N2 is
hydrogenated in three steps until the first NH3 is liberated,
and then the remaining nitrido-N is hydrogenated three more times
to yield the second NH3. In the “alternating”
(A) pathway that has been suggested to apply to catalysis at Fe of
FeMo-co,
25,131
the first two hydrogenations generate a
diazene-level intermediate, the next two form hydrazine, and the first
NH3 is liberated only by the fifth hydrogenation (Figure 8). As one can imagine alternative
structures for
the intermediates, the figure focuses on the defining difference between
D and A pathways as being the release of the first NH3 in
the D as occurring after three hydrogenations of substrate, the addition
of three [e–/H+] to substrate, but only
after five hydrogenations in A.
Figure 8
Comparison of distal (D) and alternating
(A) pathways for N2 hydrogenation, highlighting the stages
that best distinguish
them, most especially noting the different stages at which NH3(1) is released.
Simple arguments can be made for both pathways and for either
Fe
and Mo as the active site.
17,154,155,207
For example, the A route is
suggested by the fact that hydrazine is both a substrate of nitrogenase
and is released upon acid or base hydrolysis of the enzyme under turnover,
17,208−211
and is favored in computations with reaction at Fe,
131
while the D route was suggested by the fact
that until recently the only inorganic complexes that catalytically
fix N2 employ Mo and function via the D route,
24
which is computationally favored for reaction
at Mo.
139
Interestingly, this argument
is somewhat weakened by a recent study that reported small W clusters
fix N2 by the A pathway.
212
More
significantly, the argument based on N2 cleavage and catalytic
N2 fixation by Mo model complexes has lost ground by the
quite recent discovery of Fe model complexes that cleave N2 (Holland and covworkers
22,213
) and indeed that also
catalytically fix N2 (Peters and co-workers
214
).
Further support for the A pathway is
provided by considerations
of the alternative nitrogenases. It is most economical to suggest
that both the Mo-dependent nitrogenase studied here and the V-type
nitrogenase reduce N2 by the same pathway. As V-nitrogenase
produces traces of N2H4 while reducing N2 to NH3,
215
then according
to Figure 8 this enzyme can be concluded to
function via the A pathway, implying the same is true for Mo-nitrogenase.
5
Intermediates of N2 Reduction: E
n
, n ≥ 4
As can be seen
in Figure 8, characterization
of catalytic intermediates formed during the reduction of N2 could distinguish between
the D and A pathways. However, such intermediates
had long eluded capture until four intermediates associated with N2 fixation were
freeze-trapped and characterized by ENDOR spectroscopic
studies.
154,155
These four states were generated
under the hypothesis that intermediates associated with different
reduction stages could be trapped using N2 or semireduced
forms of N2 or their analogues: N2; NH=NH;
NH=N–CH3; H2N–NH2.
17,153,154
These included
a proposed early (e) stage of the reduction of N2, e(N2), obtained from wild-type
(WT) MoFe protein with N2 as substrate;
166,170
two putative “midstage”
intermediates, m(NH=N–CH3), obtained from α-195Gln MoFe protein with CH3–N=NH as substrate
170,171
and m(NH=NH), obtained from the doubly substituted,
α-70Ala/α-195Gln MoFe protein during
turnover with in-situ-generated NH=NH;
147
and a “late” stage, l(N2H4), from the α-70Ala/α-195Gln MoFe protein during turnover
with H2N-NH2
160,170
as substrate. Both hydrazine
and diazene are substrates of wild-type nitrogenase that, like N2, are reduced to
ammonia.
17,147,160,211,216
5.1
Intermediate I
A combination
of X/Q-band EPR and 15N,1,2H ENDOR measurements
on the intermediates formed with the three semireduced substrates
during turnover of the α-70Val→Ala/α-195His→Gln MoFe protein subsequently showed that
in fact
they all correspond to a common intermediate (here denoted I) in which FeMo-co binds
a substrate-derived [N
x
H
y
] moiety (Figure 9).
154−156,207
Thus, both
the diazenes and hydrazine enter and “flow through”
the normal N2-reduction pathway (Figure 8), and the diazene reduction must have “caught
up”
with the “later” hydrazine reaction.
Figure 9
Comparison of 35 GHz
ReMims pulsed 15N ENDOR spectra
of intermediates trapped during turnover of the α-70Ala/α-195Gln MoFe protein with
15N2H4, 15N2H2, and 15NH=N—CH3 (denoted 15MD).
Adapted with permission from ref (207). Copyright 2011 American Chemical Society.
1,2H and 15N 35 GHz CW and pulsed ENDOR measurements
next showed that I exists in two conformers, each with
metal ion(s) in FeMo-co having bound a single nitrogen from a substrate-derived
[N
x
H
y
] fragment.
154,155
Subsequent high-resolution 35 GHz pulsed ENDOR spectra and X-band
HYSCORE measurements showed no response from a second
nitrogen atom, and when I was trapped during turnover
with the selectively labeled CH3—15N=NH, 13CH3—N=NH, or C2H3—N=NH, no signal was seen
from the isotopic
labels.
207
From these results we concluded
the N–N bond had been cleaved in forming I, which
thus represents a late stage of nitrogen fixation, after the first
ammonia molecule already has been released and only a [NH
x
] (x = 2 or 3) fragment of substrate
is bound to FeMo-co.
207
5.2
Nitrogenase Reaction Pathway: D versus A
Given that
states that could correspond to I are reached
by both A and D pathways (Figure 8), the identity of this [NH
x
] moiety
need not in itself distinguish between pathways. However, the spectroscopic
findings about I, in conjunction with a variety of additional
considerations, led us to propose that nitrogenase functions via the
A reaction pathway of Figure 8 for reduction
of N2.
207
As one example, to
explain how nitrogenase could reduce each of the substrates, N2, N2H2, and N2H4, to
two NH3 molecules via a common A reaction pathway,
one need only postulate that each substrate “joins”
the pathway at the appropriate stage of reduction, binding to FeMo-co
that has been “activated” by accumulation of a sufficient
number of electrons (possibly with FeMo-co reorganization), and then
proceeds along that pathway. Energetic considerations,
139
in combination with the strong influence of
α-70Val substitutions of MoFe protein without modification of FeMo-co reactivity,
then implicate Fe, rather than
Mo, as the site of binding and reactivity.
146,154,217
5.3
Intermediate H
When
nitrogenase is freeze–quenched during turnover, the EPR signals
from trapped intermediates in odd-electron FeMo-co states (Kramers
states; S = 1/2, 3/2,...; E
n
, n = even),
154,155
plus the signals from residual
resting-state FeMo-co, never quantitate to the total FeMo-co present,
indicating that EPR-silent states of FeMo-co must also exist. These
silent MoFe protein states must contain FeMo-co with an even number
of electrons, and thus correspond to E
n
, n = odd (n = 2m +1, m = 0–3) intermediates in the LT scheme.
As noted above, such states may contain diamagnetic FeMo-co, or FeMo-co
in integer-spin (S = 1, 2, ...), “non-Kramers
(NK)” states,
179,180,218
but no EPR signal from an integer-spin form of FeMo-co had been
detected until careful examination of samples that contain intermediate I(154−156)
revealed an additional broad EPR signal
at low field in Q-band spectra that arises from an integer-spin system
with a ground-state non-Kramers doublet with spin S ≥ 2 (Figure 10).
219
Figure 10
2K Q-band CW EPR spectrum of α-70Val→Ala, α-195His→Gln MoFe protein in resting-state
(S = 3/2) and trapped during
turnover with 14N2H4. Kramers intermediate I and non-Kramers intermediate, H, are
noted
in the turnover spectrum. Adapted with permission from ref (219). Copyright 2012 National
Academy of Sciences.
Earlier work showed how to characterize a non-Kramers doublet
with
ESEEM spectroscopy (NK-ESEEM),
173,174
so NK-ESEEM time-waves
were collected for the NK intermediates trapped during turnover with: 14N and 15N
isotopologs of N2H2 and N2H4 substrates; 95Mo-enriched
α-70Val→Ala/α-195His→Gln MoFe protein; H—14N=14N—CH3, H—15N=14N—CH3, and H—14N=14N—CD3.
Figure 11 presents representative
35 GHz (2 K) three-pulse NK-ESEEM time-waves collected at several
relatively low fields from the nitrogenase NK intermediates generated
with isotopologs of the three substrates. The NK-ESEEM time-waves
for the intermediates trapped during turnover with the corresponding 14N and 15N isotopologues
of N2H2, N2H4, and HN2CH3 substrates are identical at all fields, indicating that
they are
associated with a common intermediate, denoted H, trapped
during turnover with all three substrates. 95Mo enrichment
of α-70Val→Ala, α-195His→Gln MoFe protein produces a significant change of the NK-ESEEM
time-wave.
This analysis established that the NK-EPR signal of H arises from the Mo-containing
FeMo-co in an integer-spin-state with S ≥ 2, and not the all-iron electron-transfer
P cluster,
also present in the MoFe protein, or even the [4Fe–4S] cluster
of the Fe protein.
219
Figure 11
Three-pulse ESEEM traces
after decay-baseline subtraction for NK
intermediate H of α-70Val→Ala, α-195His→Gln MoFe protein trapped during
turnover with 14NH=14NH, 14NH=14NCD3, 14NH2—14NH2, 15NH=15NH, 15NH=14NCH3.
Adapted with permission from ref (219). Copyright 2012 National Academy of Sciences.
Comparison of the 14N/15N NK-ESEEM of H in Figure 11 indicates that a nitrogenous
ligand derived from substrate is directly bound to FeMo-co of α-70Val→Ala/α-195His→Gln
MoFe protein.
Modulation is absent from the second 14N that would be
present if the N–N bond of substrate remained intact, as shown
by comparison of the time-waves for the H prepared with
H—15N=15N—H versus H—15N=14N—CH3, as is modulation
from 2H of H–14N=14N-CD3. This indicates that H contains an
NH
x
fragment that remains bound to FeMo-co
after cleavage of the N–N bond and loss of NH3.
Quadrupole coupling parameters for the NH
x
fragment indicated it is not NH3, and that H has bound [−NH2].
219
6
Unification of the Nitrogenase Reaction Pathway
with the LT Kinetic Scheme
The H and I intermediates provide “anchor-points”
that allow assignment of the complete set of E
n
intermediates that follow E4, 5 ≤ n ≤ 8. As illustrated in Figure 12, the loss of
two reducing equivalents and two protons as
H2 (eq 1) upon N2 binding
to the FeMo-co of E4 leaves FeMo-co activated by two reducing
equivalents and two protons. We argued that when N2 binds
to FeMo-co it is “nailed down” by prompt hydrogenation,
Figure 12, with N2 binding, H2 loss, and reduction to the diazene level, all occurring
at
the E4 kinetic stage of the LT scheme.
219
The identification of H with its E
n
stage is achieved as follows. (i) As the same intermediate H is formed during turnover
with the two diazenes and with
hydrazine, the diazenes must have catalytically “caught up”
to hydrazine, and H must occur at or after the appearance
of a hydrazine-bound intermediate. (ii) As noted above, H contains FeMo-co in an integer-spin
(NK) state, and thus corresponds
to an E
n
state with n = odd. As H is a common intermediate that contains
a bound fragment of substrate, it must, therefore, correspond to E5 or E7, and analysis
of the pathway alternatives
in the light of the EPR/ESEEM measurements indicated that H corresponds to the [NH2]−-bound
intermediate
formed subsequent to N–N bond cleavage and NH3 release
at the E7 stage of the P–A pathway.
Figure 12
Integration of LT kinetic
scheme with “prompt” (P)
alternating (A) pathway for N2 reduction. The ? represents
the product of N2 binding with H2 release, whose
identity is discussed below. Also shown is how diazene and hydrazine
join the N2 reduction pathway. Note: M denotes FeMo-co
in its entirety, and substrate-derived species are drawn to indicate
stoichiometry only, not mode of substrate binding. E
n
states, n = even, are Kramers states; n = odd are non-Kramers. MN denotes resting-state
FeMo-co. Individual charges on M and a substrate fragment, not shown,
sum to the charge on resting FeMo-co. Adapted with permission from
ref (156) with corrections
based on the re mechanism for H2 loss upon N2 binding discussed below. Copyright 2013
American Chemical Society.
By parallel arguments, the only
possible assignment for the S = 1/2 state I, which
we showed earlier to occur after N–N bond cleavage,
207
is as E8: I must correspond
to the final state in the catalytic process (Figure 12), in which the NH3 product
is bound to FeMo-co
at its resting redox state, prior to release and regeneration of the
resting-state form of the cofactor. The trapping of a product-bound
intermediate I is analogous to the trapping of a bio-organometallic
intermediate during turnover of the α-70Val→Ala MoFe protein with the alkyne, propargyl
alcohol; this intermediate
was shown to be the allyl alcohol alkene product of reduction.
168
With assignments of E4, E7, and E8, then filling in the LT “boxes”
for E5, E6 of Figure 3 is straightforward, thus unifying the reaction pathway for
N2 reduction with the LT scheme.
Figure 12 adopts a “prompt”
(P)–alternating pathway for the stages following N2 binding and H2 loss, which offers
explanations for how
the hydrogenated reaction intermediates, diazene and hydrazine, join
the N2 reduction pathway. Key to this issue was the finding
that H2 inhibits the reduction of diazene,
147
but not hydrazine.
211
We took the simplest view, that under turnover, diazene and hydrazine
each joins the N2 reduction pathway at its own characteristic
entry point, and each then proceeds to generate both H and I. As shown in Figure 12,
diazene binds to E2 with the release of H2, thereby entering the N2 pathway as the
“final”
interconverting form of the E4 state. N2H4 instead binds to E1 (as proposed for another
two-electron
substrate, C2H2
17,33,103,220
), joining the N2 pathway with the release of NH3 to form a stage
corresponding to E7 in the N2 reduction scheme.
156
7
Obligatory Evolution of H2 in Nitrogen
Fixation: Reductive Elimination of H2
The E
n
assignments of Figure 6 plus those of Figure 12 give
proposed structures to all E
n
states of
the LT kinetic scheme (Figure 3), but the assignments
have been developed through independent analyses of the two four-electron
halves of the eight-electron catalytic cycle (eq 1). In the first half (part I) of
the pathway, accumulation of four
electrons/protons activates FeMo-co, generating E4; in
the second half (part II), bound N2 is hydrogenated by
two of those electrons/protons plus an additional four electrons/protons.
However, the assignments are silent about the mechanism by which the
E4 Janus intermediate, Figure 4,
connects these two halves: the obligatory production of an H2 molecule upon N2 binding,
as shown in Figure 12.
33,156,158
Why nitrogenase should “waste” fully 25% of the ATP
required for nitrogen fixation through H2 generation (eq 1) has remained a mystery,
and indeed is not even
accepted uniformly.
23,221
Consideration of the finding
that E4 stores its four
reducing equivalents as two bridging hydrides (Figure 4) within the context of the
well-known organometallic chemistries
of hydrides
194,222
and dihydrogen
223
led us to examine the two alternative mechanisms by which
this state might bind and activate N2 with release of H2, and proceed to the prompt
formation of FeMo-co with a bound
diazene-level species (N2H2) without additional
accumulation of [e–/H+], as featured
in the P–A reaction pathway, Figure 12. In one, H2 is formed by hydride protonation
(hp mechanism),
Figure 13, upper; the other forms H2 through reductive elimination (re),
195
Figure 13, lower. We first describe these
two mechanisms, and then show that the re mechanism is operative.
Figure 13
Visualization
of hp and re mechanisms for H2 release
upon N2 (blue) binding to E4. The following
is shown: the Fe-2,3,6,7 face of resting FeMo-co; the structure of
FeMo-co must distort in different stages of catalysis. The Fe that
binds N2 is presumed to be Fe6, as indicated by studies
of α-70Val variants; when bold, red, Fe6 is formally
reduced by two equivalents (see text). The bridging hydrides of E4 (green) are positioned
to share an Fe “vertex”,
as suggested by re mechanism of H2 release upon N2 binding. Alternative binding modes
for N2-derived species
can be envisaged.
7.1
Hydride
Protonation (hp) Mechanism
In the hp scheme (Figure 13, upper), N2 binding is accompanied
by the activation of one bridging hydride to the
terminal form and protonation of this hydride
by a sulfide-bound proton to form and release H2. Such
a mechanism for H2 formation is invoked in discussions
of hydrogenases,
223,224
and there is strong precedence
for replacement of a metal-bound H2 with N2.
In this context, by analogy to the mechanism for the (much less demanding)
reduction of alkynes/alkenes one might propose that transient terminalization
of the “second” hydride would then lead to hydrogenation
and protonation of the bound N2, to form FeMo-co bound
N2H2 (see Figure 13,
upper, below). For reasons that will become clear below, the hydrogenation
of N2 to form metal bound-N2H2 must
be reversible.
7.2
Reductive Elimination (re)
Mechanism
The second mechanism for H2 loss upon
N2 binding
begins with transient terminalization of both E4 hydrides, Figure 13, lower. This
is
followed by reductive elimination of H2 as N2 binds, steps with considerable precedence.
4,194,222,223,225
Of key importance, the departing H2 carries
away only two of the four reducing equivalents stored in E4, while the Fe that binds
N2 becomes highly activated
through formal reduction by two equivalents; for example a formal
redox state of Fe(II) would be reduced to Fe(0). This delivery of
two reducing equivalents to the FeMo-co core which otherwise is reduced
by at most one equivalent during electron/proton activation (Figures 6 and 7) would
poise the cofactor
to deliver the two activating electrons to N2, whose π
acidity could be further enhanced by electrostatic interactions with
the two sulfur-bound protons: combined delivery of the two electrons
and protons would directly yield cofactor-bound N2H2. This amounts to a “push–pull”
mechanism
for the hydrogenation of N2, in which the “push”
of electrons from the doubly reduced cofactor onto N2 is
enhanced by the electrostatic “pull” of the protons
bound to sulfur. As discussed in section 9, below, models of E4(N2) constructed by
placing
N2 and two protons on the doubly reduced FeMo-co core modeled
with its resting-state structure provide a convincing illustration
of this mechanism (and other insights, as well). The diminished electron
donation to Fe by protonated sulfides would not only facilitate reductive
elimination, but also would act to localize the added electrons on
the Fe involved, limiting charge delocalization over the rest of the
cofactor. This mechanism provides a compelling rationale for obligatory
H2 formation during N2 reduction: the transient
formation of a state in which an electrostatically activated N2 is bound to a highly
activated, doubly reduced site, thereby
generating a state optimally activated to carry out the initial hydrogenations
of N2, the most difficult process in N2 fixation.
7.3
Mechanistic Constraints Reveal That Nitrogenase
Follows the re Mechanisms
A clear choice between hp and re
mechanisms is achieved by testing them against the numerous constraints
that are associated with the reaction of D2 with the diazene-level
E4(N2/N2H2) state formed
when N2 binds to the cofactor and is reduced. The three
principal constraints are listed in Chart 1. The first test that they provide for
a mechanism is that it must
accommodate the finding that when nitrogenase turns over in the presence
of both N2 and D2, then two
HD are formed through D2 cleavage and solvent-proton reduction,
with the stoichiometry summarized as constraint i of Chart 1.
17,226−228
Such HD formation only occurs in the presence of
N2, and not during reduction of H+ or any other substrate.
226,229,230
Chart 1
The second key constraint and mechanistic test was revealed
by
Burgess and co-workers 30 years ago; the absence of exchange into
solvent of D+/T+ derived from D2/T2 gas, Chart 1, constraint ii.
226
When nitrogenase turns over under a mixture
of N2 and T2, HT is formed with stoichiometry
corresponding to Chart 1, constraint i, but during this process only a negligible
amount of T+ is released to solvent (∼2%). The third constraint
is provided by a later study of α-195His- and α-191Gln-substituted MoFe proteins.
161
It provided persuasive evidence that HD formation under N2/D2 requires that the enzyme
be at least at the E4 redox level, with a FeMo-co-bound N–N species at the
reduction level of N2H2 or beyond, corresponding
to the third constraint, Chart 1, iii.
161
Constraint iii, plus the stoichiometry of HD
formation according to constraint i implies a process described as
3
Thus, N2H2 formation
is reversible, as shown in Figure 13.
Figure 14, upper, shows that the characteristics
of HD formation during turnover under N2/D2
cannot be reconciled with the hp mechanism. In the reverse
of this mechanism, D2 binding and N2 release
would generate an E4 state that has one deuteride bridge,
which is deactivated for exchange with solvent. However, it carries
the other deuteron in the form of D+ bound to sulfur (or
a protein residue), which likely would be solvent exchangeable. Exchange
of that D+ would violate the stoichiometric constraint,
of eq 3 (line i, Chart 1), as relaxation of E4 to E2 within the reverse-hp
mechanism would generate only one HD per D2, not two as
required. Correspondingly, replacement of D2 by T2 in Figure 14, upper, with exchange
of T+ bound to sulfide would lose roughly one T+ per
T2 to solvent, contrary to the few percent loss observed
by Burgess et al. (constraint ii).
226
The possibility that the proton-bearing site is “shielded”
from exchange seems implausible for a catalytic cluster that depends
on proton delivery for its catalytic function, and in any case solvent
exchange need not be fast; the rate-limiting step in nitrogenase turnover
is the off-rate for Fe protein after it has delivered its electron
to MoFe,
33,99
and this process is quite slow, with a rate
constant of ∼6 s–1.
Figure 14
Reversal of hp and re
mechanisms upon D2 binding. Details
as in Figure 13. Bold arrows replace equilibrium
arrows to emphasize the relaxation process.
If this proton were nonetheless shielded from exchange, relaxation
to E2 would occur with regeneration of D2, without
the generation of HD, in disagreement with the stoichiometric constraint
of Chart 1. This objection would be overcome
if at ambient temperatures the hydrides/protons can “migrate”
over the FeMo-co face, but this instead would require multiple sites
to be “shielded” for slow exchange, while FeMo-co is
accessible to rapid proton delivery. Overall, we conclude that the
hp process fails to satisfy the constraints of Chart 1, as the reverse hp process
satisfies neither the stoichiometry of eq 3 nor the constraint
that T+ is not released to solvent (Chart 1).
In contrast, in the reverse of the re mechanism,
shown in Figure 14, lower, D2 binding
and N2 release would generate E4(2D), the E4 isotopomer
in which both atoms of D2 exist as deuteride
bridges. This state would relax with loss of HD to E2(D),
and then to E0 with loss of the second HD, thus satisfying
the stoichiometry of eq 3. If the reaction were
carried out under T2, essentially no T+ would
be lost to solvent because the bridging deuterides are deactivated
for exchange with the protein environment and solvent, thus satisfying
the “T+ exchange” constraint, Chart 1.
One alternative fate of the E4(2D) formed by D2 replacement of N2 would be
to rebind an N2, but this would merely release the D2 that had started
the reverse process, creating a cycle invisible to detection. As a
second alternative, E2(D) could acquire two additional
electrons/protons to achieve the monodeutero E4 state.
However, as shown in Figure 14, lower, if this
state then bound N2 it would release the second HD, again
without solvent exchange, whereas if it ultimately relaxed to E0 it would release
the second HD along with an H2. Thus, the re mechanism for N2 binding and H2 release
not only has the compelling chemical rationale discussed
above, but also satisfies the three critical HD constraints for the
various alternatives that arise when it is run in reverse, Chart 1.
In short, the re mechanism, Figure 13, lower,
satisfies the constraints summarized in Chart 1, as visualized in Figure 14, lower;
to the
best of our knowledge, it likewise satisfies all other constraints
on the mechanism provided by earlier studies, most of which are not
directly tied to D2 binding.
8
Test of
the re Mechanism
Subsequent to formulation of the re mechanism
for the activation
of FeMo-cofactor to reduce N2 (Figure 13, lower),
156
we noted that addition
of C2H2 to a N2/D2 reaction
mixture should offer a rigorous test of the mechanism. The test is
founded on a defining characteristic of nitrogenase catalysis, an
exact distinction between hydrons (H/D/T) associated with the gaseous
diatomics, H2/D2/T2, and those derived
from solvent water. Thus, when nitrogenase in protic buffer is turned
over under N2/D2, gaseous D2 can
displace N2 from the E4(N2/N2H2) state (Figure 14, lower),
stoichiometrically yielding two HD.
226−228
This and other observations
clearly show that diatomic H2/D2 is not used
to reduce N2 during turnover under N2//H2/D2 (in particular, T incorporated into the
ammonia
product of N2 fixation would exchange with solvent).
17
Likewise, as demonstrated below, when C2H2 is reduced in the presence of D2,
no deuterated ethylenes are generated.
8.1
Predictions
With this foundation,
we recognized that the re mechanism predicts that turnover under C2H2/D2/N2 should
not only
incorporate H from solvent to generate C2H4 by
the normal reduction process, but through the agency of the added
N2 also should breach the separation of gaseous D2 from solvent protons by generating
both C2H3D and C2H2D2 (Figure 15). According to the re mechanism, when turnover
is carried out under N2/D2, D2 can
react with E4(N2/N2H2),
replacing the N2 and undergoing oxidative addition to generate
E4(2D). We recognized that this state in fact might be
expected to react with C2H2 to form C2H2D2 through the idealized mechanism (Figure
16A) involving terminalization of an [Fe–D–Fe]
bridge of E4, and migratory insertion of bound C2H2 into the Fe–D bond to form an
Fe-alkenyl intermediate,
followed by reductive elimination of C2H2D2.
195,231
Previous studies
17,103
could not distinguish reaction at the E4 state from reaction
at the E2 state when C2H2 is reduced
in the absence of N2, as N2 is required to enable
gaseous D2 to enter the nitrogenase catalytic process.
The possibility that acetylene can access different nitrogenase redox
states, however, had been suggested on the basis of experiments using
a nitrogenase variant that exhibits N2 reduction that is
resistant to inhibition by acetylene.
232,233
Figure 15
Formation
of deuterated acetylenes during turnover under N2/D2/C2H2 as predicted according
to re mechanism. Cartoons again depict the Fe2,3,6,7 face of resting-state
FeMo-co, with no attempt to incorporate likely structural modifications.
Figure shows that the “reverse” of re mechanism through
displacement of N2 by D2 produces, successively,
E4(2D) and E2(D), further showing potential
reaction channels for capture of E4(2D) and E2(D) intermediates with C2H2.
Figure 16
Schematic mechanism for reaction of C2H2 with
E4(2D) and E2(D). (A) Formation of C2H2D2, which follows Scheme 15.20 of Hartwig:
231
mi = migratory insertion; re = reductive elimination.
In braces: Possible alternative reaction channel that leads to formation
of C2H3D, ap = alkenyl protonolysis. (B) Schematic
mechanism for formation of C2H3D from reaction
of C2H2 with E2(D). (C) Illustration
of possibility that C2H2 displaces D2 formed by reductive elimination of the E4(2D)
deuterides,
leading to direct formation of C2H4 without
D incorporation.
The E4(2D)
state also would relax through the loss of
HD to form E2(D), an E2 state whose unique isotopic
composition can be generated in no other way. Interception of the
E2(D) state by C2H2 would then generate
C2H3D, with Figure 16B presenting a plausible mechanism: deuteride terminalization
and
insertion, followed by alkenyl protonolysis.
195,231
This reaction also might occur through an alternative reaction channel
of E4(2D), as noted in Figure 15.
8.2
Testing the Predictions
We tested
the predictions based on the re mechanism of an unprecedented involvement
of gaseous D2 in substrate reduction by use of C2H2 reduction under N2/D2/C2H2 gas
mixtures to intercept the E4(2D) and
E2(D) states. As expected, the control reaction of turnover
under D2/C2H2 generates only C2H4, without incorporation of D from gaseous D2 to generate
either C2H3D or C2H2D2 (Figure 17). In
dramatic contrast, C2H2 reduction by nitrogenase
under a N2/D2/C2H2 gas
mixture in fact produces readily measured amounts of C2H2D2 and even greater amounts
of C2H3D (Figure 17).
157
Figure 17
Time-dependent formation of 13C2H3D and 13C2H2D2, catalyzed
by nitrogenase reduction of 13C2H2. 13C2H3D determined by GC/MS monitoring
of m/z = 31 for a reaction mixture
containing 13C2H2 and including D2 and N2 (■), just D2 (x inside
□), or H2 and N2 (□). Inset: 13C2H2D2, m/z = 32, formation starting with 13C2H2/D2/N2
(●), just
D2 (◊), or H2/N2 (○).
Partial pressures of 0.02 atm 13C2H2, 0.25 atm N2, and 0.7 atm H2/D2, where present.
The molar ratio of Fe protein to MoFe protein was
2:1. All assays incubated at 30 °C. Adapted with permission from
ref (157). Copyright
2013 National Academy of Sciences.
On reflection, the success of this test for the formation
of E4(2D) is a consequence of the greater reactivity of
C2H2 compared to that of N2 and/or
of the difference
in the likely ways that these two substrates bind to FeMo-co: side-on
for C2H2, end-on for N2. Otherwise,
in a process analogous to that for N2H2 formation
in the re mechanism (Figure 13, lower), C2H2 might in principle displace D2 formed
by reductive elimination of the E4(2D) deuterides, leading
to direct formation of C2H4 without D incorporation,
Figure 16C. The yield of C2H2D2 may be less than that of C2H3D, because the contribution
from this reaction channel diminishes
the yield of the former, but it is perhaps more likely that the binding
and reduction of C2H2 by E4(2D) is
substantially less likely than the relaxation of E4(2D)
to E2(D) through loss of HD, and the reduction of C2H2 by E2(D) (Figure 15).
These observations are enriched by consideration
of the dependences
of the yields of C2H3D and C2H2D2 on the partial pressures of C2H2, D2, N2, and electron
flux, all of
which are understandable in terms of the production of the E4(2D) and E2(D) states
under these turnover conditions,
as predicted by the re mechanism for FeMo-cofactor activation for
N2 binding and reduction.
157
For example, reduction of acetylene and N2 are mutually
exclusive, with complicated inhibition kinetics between these two
substrates.
217,234
Therefore, it was of interest
to determine the effect of varying the N2 partial pressure
on the formation of C2H3D and C2H2D2 at fixed C2H2 and D2 pressures. The yields of
C2H3D and
C2H2D2 increase in parallel with
increasing partial pressure of N2 (Figure 18). This can be explained by enhanced formation
of E4[N2/N2H2] by reaction of N2 with E4. Increased formation of E4[N2/N2H2] in turn
would enhance reaction with
D2 to form E4(2D), which can be intercepted
by acetylene to form deuterated ethylenes (Figure 15).
157
Figure 18
Deuterated ethylene
formation as a function of N2 partial
pressure. The partial pressure of C2H2 was 0.02
atm and D2 was 0.6 atm. The molar ratio of Fe protein to
MoFe protein was 4:1. Assay conditions as in Figure 17. Adapted with permission from
ref (157). Copyright 2013 National Academy of Sciences.
It is of interest to note that
the reduction of C2H2 to C2H3D by reaction with E2(D) formally corresponds to
the reduction of C2H2 by the HD that otherwise
would form during relaxation of E2(D) to E0,
a perspective that highlights the contrast
between this result, achieved in the presence of N2, with
the failure of nitrogenase to use H2/D2 to reduce
any substrate in the absence of N2. As an eleboration on
this perspective, the formation of HD during turnover under N2/D2, with stoichiometry
(eq 3, above),
17
can be seen to correspond
to the nitrogenase-catalyzed reduction of protons by D2 and electrons with N2 as cocatalyst,
eq 3′
3′
as the reaction neither proceeds without N2 nor consumes
N2. Likewise, although C2H2D2 is well-known to form during nitrogenase
reduction of C2D2 in H2O buffer (or
C2H2 in D2O buffer),
148
formation of this species during turnover under
C2H2/D2/N2 corresponds
to the previously unobserved reduction of C2H2 by gaseous D2 with N2 as cocatalyst
(eq 4).
4
Correspondingly,
the formation of C2H3D involves incorporation
of D– derived
from D2 along with H+ from solvent with N2 as cocatalyst.
9
Completing the Mechanism
of Nitrogen Fixation
Figure 12, above,
presents a formal integration
of the reaction pathway for nitrogen fixation (intermediates E4–E8) with the LT kinetic
scheme, the key
to the resulting mechanism being N2 binding and H2 release through the re mechanism,
Figure 13, lower. This mechanism is built on the structure of the E4 intermediate
and its implication that hydride chemistry is central
to nitrogen fixation by nitrogenase (section 3). As a corresponding implication, we
further offered the two alternative
sets of proposed structures for the “early”, E1–E3, intermediates (Figure 6). We now
discuss in greater depth the E5–E8 intermediates of nitrogen fixation, proposing in
Figure 19, II not only more detailed structures for the
stages following the formation of N2H2-bound
FeMo-co, the E4(N2H2) state, written
as binding diazene itself, but also the nature of the chemical transformations
that link these stages during the delivery of the ‘second half’
of the eight [e–/H+] that comprise the
stoichiometry of nitrogen fixation, eq 1. The
analysis further leads us to provisionally assign the early, “first
half” intermediates to the alternative described in Figure 6B, now visualized in Figure
19, I. When combined with the reductive elimination (re) mechanism
for the binding N2 and release of H2, Figure 13, lower, the result, Figure 19, is
a self-consistent proposal for the structures of all intermediates
in the nitrogen fixation mechanism and a formal description of the
transformations that convert each stage to the subsequent one: a complete,
though of course still simplified, mechanism for nitrogen fixation
by nitrogenase.
Figure 19
Proposed mechanism displaying structures of all intermediates
in
nitrogen fixation, inspired by the assumption of primacy of hydride
chemistry associated with the Fe2,3,6,7 face of FeMo-co, and containing
a formal description of the transformations that convert each stage
to the subsequent one. In I the mechanism tentatively adopts and visualizes
the view of E
n
states n = 1–4 presented in Figure 6B; in II
it visualizes bridging hydrides by analogy, without evidence for or
against terminal hydrides for n = 5–7. Likewise,
the structure of the N2H2 species as end-on
bound diazene is suggestive, not definitive, etc. I and II are connected
by the re mechanism, Figure 13, lower. Formal
charges are included as useful to help guide the reader.
Figure 19, II, is constructed
on two assumptions
that (i) the formation and reactions of hydrides is key; (ii) beginning
with N2H2, the hydrogenation of reduced forms
of N2 involves migratory insertion into Fe–H bonds.
These assumptions lead to the conclusion that [e–/H+] transfer to FeMo-co of the E4(N2H2)
and E6 states creates E5 and
E7 that each contain an [Fe–H–Fe] bridging
hydride moiety bound to an oxidized FeMo-co, Figure 19, II, in correspondence with
the analogous [e–/H+] transfer to E0 and E2, shown in the cartoon of Figure 6B,
and now visualized in Figure 19, I. In the
case of E5, an accompanying migratory insertion of the
N2H2 into an Fe–H bond (presumably formed
by terminalization of the bridge) forms the [N2H3]− moiety bound to the oxidized cluster;
in the
case of E7, migratory insertion leads to N–N bond
cleavage and formation of [NH2]− bound
to the formally oxidized cofactor (Figure 19, II). The follow-up [e–/H+] transfer
to E5, E7 generates the E6 and E8 states, respectively. This mechanistic
picture is anchored by the final stages, E7 and E8, whose structures match those proposed
in the EPR/ENDOR/ESEEM studies
of intermediates H, assigned to E7, and I, assigned to E8, Figure 12.
The proposal completes a mechanism in which the stoichiometrically
required delivery of all 8 [e–/H+] to
FeMo-co is controlled by the hydride chemistry of the cofactor. The
clearly understandable differences between the first “half”
of the catalytic cycle, visualized in Figure 19, I, and the “second half”, Figure
19, II, arise because the former involves accumulation of reducing
equivalents while the latter involves delivery of reducing equivalents
to substrate.
The two halves are similar in that addition of
[e–/H+] to form an n = odd intermediate
(n = 1, 3, first half; n = 5, 7,
second half) generates an [Fe–H–Fe] bridging hydride
attached to a formally oxidized FeMo-co core. They differ in that
the hydride is “stored” in n = 1, 3,
but is promptly transferred to substrate in n = 5,
7 to form a (formally) anionic reduced substrate. Upon addition of
[e–/H+] to any one of these four n = odd intermediates, to form the subsequent n =
even intermediate, the electron formally reduces the core to the
resting-state redox level. In the first half (n =
2, 4), the H+ is delivered to a sulfur and its charge balances
that on a hydride; in the second half (n = 6, 8),
the proton neutralizes the anionic nitrogenous ligand, to form the
neutral, N2H4 of E6, NH3 of E8.
The two halves of the nitrogen fixation
mechanism are joined at
the E4 stage, as described above and displayed as Figure 19, re: the E4(2H) intermediate
formed
by accumulation of four [e–/H+] and containing
two bridging hydrides undergoes reductive elimination as it binds
N2 and releases the two “sacrificial” reducing
equivalents as H2. Figure 19 thus
represents a complete mechanism for nitrogen fixation by nitrogenase
that invokes the primacy of the hydride chemistry of FeMo-co.
9.1
Uniqueness of N2 and Nitrogenase
The mechanistic
proposal of Figure 19 invokes
the primacy of hydride chemistry associated with a 4Fe face of FeMo-co,
a structural feature made possible only with a cluster of at least
six metal ions. The hydrogenations of reduced forms of N2, starting with N2H2, involve
migratory insertion
of substrate into Fe–H bonds, one at a time. This is the same
mechanism visualized for the “normal” reduction of C2H2 at the E2 stage, and even
for the
rare trapping of E4 by C2H2, Figure 16; we suggest migratory insertions are likely
to
be involved in the hydrogenation of all other substrates.
But
N2 is not reactive to hydride insertion. So nitrogenase
adopts a different “strategy” for attacking its physiological
substrate. It is forced to accumulate four reducing equivalents as
two Fe hydrides, which requires a 4-Fe face, and thus the large cluster
is “held together” by the carbide at its core. We have
concluded that this cluster can only become activated for N2 hydrogenation through
reductive elimination of two of those equivalents
in the form of H2.
156,157
The “push”
of the doubly reduced metal-ion core of the cluster, compounded by
the electrostatic “pull” of sulfur-bound protons, is
required to overcome the high barrier to the initial hydrogenation
of N2, directly to N2H2, Figure 19.
9.2
Structure of the E4(N2) Intermediate: Some Implications
As
an exercise to illustrate
four points worth noting, we have modeled alternative structures of
the E4(N2) intermediate by building the bound
substrate onto the crystal structure of resting-state FeMo-co using
structural information from model complexes.
235−240
It seems most likely, on the basis of the structures of model complexes,
that N2 binds end-on, rather than bridging. As illustrated
in Figure 20, and emphasized over the years,
137,241,242
end-on bound N2 can
bind to FeMo-co in two basic, alternative modes: endo, with the N2 “nestled” in the
pocket above
the Fe2,3,6,7 face; exo, with N2 pointed
away from that face. The first point is as follows. According to our
mechanism, E4(N2) contains doubly reduced metal-ion
core with two protons bound to sulfur. There are multiple potential
dispositions of the H+ on different sulfurs, but distance
measurements with the mockups show that the atoms of N2 and protons can indeed be
in close enough proximity to support the
electrostatic “pull” postulated above.
Figure 20
Models for the two alternative
modes for N2 binding
at Fe6 of FeMo-cofactor in the E4(N2) state,
with two protons bound to two adjacent sulfides as in Figure 4: (A) endo mode; (B)
exo mode. The side chains of selected amino acid residues are shown
as sticks. The figure was generated in Pymol by building N2 onto the resting-state
of FeMo-co using the coordinate file PDB:2AFK. Iron is shown in
rust, molybdenum in magenta, sulfur in yellow, carbon in dark gray,
hydrogen in light gray, nitrogen in blue, and oxygen in red.
Second, this mockup demonstrates
the commonly understood need for
the FeMo-co core to “relax” upon substrate binding.
In the resting-state the Fe ions are roughly tetrahedral, and without
such relaxation, the N2–S distances would be far
too short. The normal assumption would be that Fe6 roughly forms a
plane with three S atoms, with a major contribution to the relaxation
being an elongation of the bond trans to N2.
The third issue is the resulting structural/electronic-structure
consequences of the identity of the trans ligand in exo versus endo N2 binding, and
it does not appear to have been widely discussed.
The modulation of metal-ion reactivity by variations in the trans ligand (the “trans
effect”)
is well-known,
231
and recently, a series
of trigonal Fe complexes that are biomimetic of nitrogenase have shown
that the trans ligands to a terminal Fe–N2 can regulate the ability of the complex
to catalytically
reduce N2.
214
In the exo binding mode, the interstitial carbide is trans to N2. This mode would
favor the idea that carbide modulates the
properties of Fe6 through the trans effect, and may
well act as a hemilabile ligand. However, in the endo mode, which has been favored
by some computations, the trans ligand is now a S that bridges to Mo. As C is (roughly)
an “in-plane”
ligand, not trans, its influence on reactivity would
be different than for endo binding, in which case
modulation of Fe6 reactivity by the trans effect
would involve [S–Mo] being “axial” ligand.
There is a corollary to considerations of the endo binding mode. It is widely assumed
that the catalytic centers of
the alternative nitrogenases have the same structure as FeMo-co, with
the heterometal atom Mo being replaced by V or Fe.
18
Thus, if N2 does bind endo to
Fe6 of a FeMo-co-like structure in all three systems, its reactivity
would be modulated by differences in the axial −S–M
“ligand” caused by differences in the properties of
Mo, V, and Fe.
The fourth point is the possible importance of
interactions of
substrate with adjacent amino acid residues. In the endo binding mode the N2 is nestled
within a binding pocket
capped by the side-chain of α-70Val; in the exo mode, the N2 is pointed to a pocket
surrounded
by α-191Gln and the homocitrate ligand of Mo. The
present mockups suggest that the protein environment in either binding
mode could readily accommodate, and even stabilize, N2 with
no more than minor conformational rearrangements.
10
Summary of Mechanistic Insights
10.1
Catalytic
Intermediates of N2 Fixation
Two major points
can be made regarding intermediates trapped: (1)
Characterization of the E4 “Janus” intermediate
as bearing four reducing equivalents in the form of two [Fe–H–Fe]
bridging hydrides has provided the foundation for proposals that the
FeMo-co core is never oxidized or reduced by more than one equivalent
relative to the resting-state, and that the oxidative couple in fact
is operative, Figure 19, I. (2) The characterization
of the common intermediates H and I, trapped
during turnover with nitrogenous substrates, led to the proposed unification
of kinetic scheme and A reaction pathway, Figure 12.
10.2
re Mechanism
Reductive elimination
of two hydrides upon N2 binding (re mechanism) provides
an explanation for the nitrogenase stoichiometry (eq 1) and for the obligatory formation
of H2 upon N2 binding. This mechanism for H2 production upon
N2 binding to E4, Figure 13, lower, satisfies both the stoichiometric constraint of
HD formation
(Chart 1, line i) and the “T+” constraint against exchange of gas-derived hydrons
with
solvent (Chart 1, line ii), whereas the hp
mechanism (Figure 13, upper) satisfies neither.
The re mechanism further involves D2 binding to a state
at the “diazene level” of reduction, as required by
the constraint of eq 3 and Chart 1, line iii. Finally, to the best of our knowledge,
all other
constraints on the mechanism, most of which are not directly tied
to D2 binding, are satisfied, as well.
This mechanism
answers the following long-standing and oft-repeated question: Why
does nature “waste” four ATP/two reducing equivalents
through an obligatory loss of H2 when N2 binds?
The answer follows: reductive elimination of H2 upon binding
of N2 to FeMo-co of the E4 state generates a
state in which highly reduced FeMo-co binds N2, which likely
is activated for reduction through electrostatic interactions with
the remaining two sulfur-bound protons. Transfer of the two reducing
equivalents generated by the reductive elimination, combined with
transfer of the two activating protons, then forms N2H2, Figure 13, lower, in keeping
with
the P–A scheme of Figure 12. It appears
that only through this activation is the enzyme able to hydrogenate
N2.
10.3
Turnover under N2/D2/C2H2 as a Test of the re Mechanism
This mechanism has been supported by a rigorous test which provided
experiments in which C2H2 is added to an N2/D2 reaction mixture. Although diatomic
D2 does not reduce nitrogenase C2H2 in the absence
of N2, the re mechanism successfully predicted that turnover
under C2H2/D2/N2 would
breach the separation of gaseous D2 from solvent protons
by generating both C2H3D and
C2H2D2.
The conclusions regarding
H2 formation upon N2 binding reached from this
study are as follows. (i) The unprecedented incorporation of D from
D2 into the nitrogenase reduction products C2H2D2 and C2H3D during
turnover under C2H2/D2/N2 in H2O demonstrates the presence of the E4(2D) and E2(D)
states under these conditions. In our view
any model that fails to incorporate obligatory H2 loss
as a fundamental aspect of N2 activation is unlikely to
provide a robust description of the chemistry associated with the
biological process.
242
(ii) This incorporation
provides a very clear demonstration of the essential mechanistic role
for obligatory, reversible loss of H2 upon N2 binding and thus of the eight-electron
stoichiometry for nitrogen
fixation by nitrogenase embodied in eq 1. Until
now, the data indicating that some H2 must be evolved during
N2 reduction has been viewed as being much more compelling
than the data indicating an obligatory evolution of one H2 for every N2 reduced, leading
to the stoichiometry of
eq 1.
17
(iii) The
formation of E4(2D) and E2(D) during turnover
under D2/N2 in H2O is predicted by
the re mechanism for the activation of FeMo-cofactor for reduction
of N2, and the interception of these intermediates by C2H2 thus provides direct experimental
evidence in
support of this mechanism (Figure 17). (iv)
The well-known reduction of protons by D2 to form 2HD during
turnover under D2/N2 in H2O and the
newly discovered reductions of C2H2 by D2/N2 should be viewed as being catalyzed by
nitrogenase
with N2 as cocatalyst. (v) This review has proposed an
explanation of the inability of H2/D2 to reduce
nitrogenase and/or catalyze substrate reduction in the absence of
N2.
11
Conclusions
The
trapping and characterization of five nitrogenase catalytic
intermediates, which correspond to three of the five stages involved
in binding and reduction of nitrogen (Figure 3), most especially the “Janus intermediate”,
E4, and including the nitrogenous intermediate states H (E7) and I (E8), have
identified the “prompt–alternating (P–A)”
pathway of Figure 12, carried out on a four-Fe
face of FeMo-co, as most likely operative for nitrogenase and led
to the unification of the nitrogenase reaction pathway and the LT
kinetic scheme.
The recognition of the central role played by
conversion of accumulated
[e–/H+] into metal hydrides has led to
the proposal that this most complex of biological catalytic clusters,
and by extension perhaps all biological clusters involved in multielectron
substrate hydrogenation, function through a limited set of redox couples,
and indeed most likely through a single couple, with multiple reducing
equivalents being stored as hydrides rather than as reduced metal
ions, Figures 6, 19.
Only in this way can a cluster accumulate equivalents delivered at
a constant potential set by its biological partners. These considerations
provide part of the reason why such a large cluster is required for
nitrogenase catalysis.
Simple energetic considerations have
further illuminated the heretofore
puzzling observation that states of nitrogenase activated by the accumulation
of multiple [e–/H+] can relax through
release of H2 (Scheme 1), but H2 cannot reduce nitrogenase in what appears to be the
reverse
process: the answer is that the processes are not microscopic reverses.
Perhaps the central question of nitrogen fixation by nitrogenase
has been that of stoichiometry: “Why does (or even, does) nature ‘waste’ four ATP/two
reducing equivalents
through an obligatory loss of H2 when N2 binds?”
An answer has been proposed on the basis of further consideration
of hydride chemistry exhibited by E4: the enzyme exhibits
the stoichiometry of eq 1 because reductive
elimination (re) of two [e–/H+] in the
form of H2 activates FeMo-co for hydrogenation of N2 to N2H2 via a “push–pull”
mechanism, Figure 13, lower. A test of the
mechanism involving turnover under N2/D2/C2H2, as in Figure 15, validated
the re mechanism, and in so doing confirmed the stoichiometry of nitrogen
fixation, eq 1, as requiring eight [e–/H+].
The test reaction further highlighted the
role of N2 as cocatalyst in reductions catalyzed by nitrogenase
that would
not occur in the absence of N2. We have further noted some
issues regarding the uniqueness of N2 and nitrogenase as
the catalyst for its hydrogenation, and of the implications of alternative
structures of the N2 complex.
The result of these
efforts is the mechanism for nitrogen fixation
presented in Figure 19 for further tests, both
experimental and theoretical.