The Nitric
Oxide Reductase Mechanism of a Flavo-Diiron
Protein: Identification of Active-Site Intermediates and Products

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Abstract

The unique active site of flavo-diiron proteins (FDPs) consists of a nonheme diiron-carboxylate site proximal to a flavin mononucleotide (FMN) cofactor. FDPs serve as the terminal components for reductive scavenging of dioxygen or nitric oxide to combat oxidative or nitrosative stress in bacteria, archaea, and some protozoan parasites. Nitric oxide is reduced to nitrous oxide by the four-electron reduced (FMNH ₂–Fe ^IIFe ^II) active site. In order to clarify the nitric oxide reductase mechanism, we undertook a multispectroscopic presteady-state investigation, including the first Mössbauer spectroscopic characterization of diiron redox intermediates in FDPs. A new transient intermediate was detected and determined to be an antiferromagnetically coupled diferrous-dinitrosyl ( S = 0, [{FeNO} ⁷] ₂) species. This species has an exchange energy, J ≥ 40 cm ^–1 ( J S ₁ ° S ₂), which is consistent with a hydroxo or oxo bridge between the two irons. The results show that the nitric oxide reductase reaction proceeds through successive formation of diferrous-mononitrosyl ( S = ¹/ ₂, Fe ^II{FeNO} ⁷) and the S = 0 diferrous-dinitrosyl species. In the rate-determining process, the diferrous-dinitrosyl converts to diferric (Fe ^IIIFe ^III) and by inference N ₂O. The proximal FMNH ₂ then rapidly rereduces the diferric site to diferrous (Fe ^IIFe ^II), which can undergo a second 2NO → N ₂O turnover. This pathway is consistent with previous results on the same deflavinated and flavinated FDP, which detected N ₂O as a product ( HayashiBiochemistry2010, 49, 7040[ PubMed]). Our results do not support other proposed mechanisms, which proceed either via “super-reduction” of [{FeNO} ⁷] ₂ by FMNH ₂ or through Fe ^II{FeNO} ⁷ directly to a diferric-hyponitrite intermediate. The results indicate that an S = 0 [{FeNO} ⁷}] ₂ complex is a proximal precursor to N–N bond formation and N–O bond cleavage to give N ₂O and that this conversion can occur without redox participation of the FMN cofactor.

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Hyperfine-Shifted 13C and 15N NMR Signals from Clostridium pasteurianum Rubredoxin: Extensive Assignments and Quantum Chemical Verification

I-Jin Lin, Bin Xia, David King … (2009)

Stable isotope-labeling methods, coupled with novel techniques for detecting fast-relaxing NMR signals, now permit detailed investigations of paramagnetic centers of metalloproteins. We have utilized these advances to carry out comprehensive assignments of the hyperfine-shifted 13C and 15N signals of the rubredoxin from Clostridium pasteurianum (CpRd) in both its oxidized and reduced states. We used residue-specific labeling (by chemical synthesis) and residue-type-selective labeling (by biosynthesis) to assign signals detected by one-dimensional 15N NMR spectroscopy, to nitrogen atoms near the iron center. We refined and extended these 15N assignments to the adjacent carbonyl carbons by means of one-dimensional 13C[15N] decoupling difference experiments. We collected paramagnetic-optimized SuperWEFT 13C[13C] constant time COSY (SW-CT-COSY) data to complete the assignment of 13C signals of reduced CpRd. By following these 13C signals as the protein was gradually oxidized, we transferred these assignments to carbons in the oxidized state. We have compared these assignments with hyperfine chemical shifts calculated from available X-ray structures of CpRd in its oxidized and reduced forms. The results allow the evaluation of the X-ray structural models as representative of the solution structure of the protein, and they provide a framework for future investigation of the active site of this protein. The methods developed here should be applicable to other proteins that contain a paramagnetic center with high spin and slow electron exchange.