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      Cadmium inhibitory action leads to changes in structure of ferredoxin:NADP + oxidoreductase

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          Abstract

          This study deals with the influence of cadmium on the structure and function of ferredoxin:NADP + oxidoreductase (FNR), one of the key photosynthetic enzymes. We describe changes in the secondary and tertiary structure of the enzyme upon the action of metal ions using circular dichroism measurements, Fourier transform infrared spectroscopy and fluorometry, both steady-state and time resolved. The decrease in FNR activity corresponds to a gentle unfolding of the protein, caused mostly by a nonspecific binding of metal ions to multiple sites all over the enzyme molecule. The final inhibition event is most probably related to a bond created between cadmium and cysteine in close proximity to the FNR active center. As a result, the flavin cofactor is released. The cadmium effect is compared to changes related to ionic strength and other ions known to interact with cysteine. The complete molecular mechanism of FNR inhibition by heavy metals is discussed.

          Electronic supplementary material The online version of this article (doi:10.1007/s10867-012-9262-z) contains supplementary material, which is available to authorized users.

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          Mechanisms of tryptophan fluorescence shifts in proteins.

          Tryptophan fluorescence wavelength is widely used as a tool to monitor changes in proteins and to make inferences regarding local structure and dynamics. We have predicted the fluorescence wavelengths of 19 tryptophans in 16 proteins, starting with crystal structures and using a hybrid quantum mechanical-classical molecular dynamics method with the assumption that only electrostatic interactions of the tryptophan ring electron density with the surrounding protein and solvent affect the transition energy. With only one adjustable parameter, the scaling of the quantum mechanical atomic charges as seen by the protein/solvent environment, the mean absolute deviation between predicted and observed fluorescence maximum wavelength is 6 nm. The modeling of electrostatic interactions, including hydration, in proteins is vital to understanding function and structure, and this study helps to assess the effectiveness of current electrostatic models.
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            Secondary structure and dosage of soluble and membrane proteins by attenuated total reflection Fourier-transform infrared spectroscopy on hydrated films.

            Attenuated total reflection Fourier-transform infrared spectroscopy of thin hydrated films of soluble and membrane protein included in a phospholipid bilayer is shown to provide useful information as to the secondary structure of the protein. The analysis of the amide I band of deuterated samples by Fourier self-deconvolution followed by a curve fitting was performed by a new procedure in which all the input parameters are generated by the computer rather than by the investigator. The results of this analysis provide a correct estimation of the alpha-helix and beta-sheet structure content with a standard deviation of 8.6% when X-ray structures are taken as a reference. We also show that the orientation of the different secondary structures resolved by the Fourier self-deconvolution/curve-fitting procedure and of the phospholipid acyl chains can be simultaneously evaluated for membrane proteins reconstituted in a lipid bilayer. Of special interest for reconstitution of membrane proteins, the lipid/protein ratio can be accurately and quickly determined from the infrared spectrum.
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              Open questions in ferredoxin-NADP+ reductase catalytic mechanism.

              Ferredoxin (flavodoxin)-NADP(H) reductases (FNR) are ubiquitous flavoenzymes that deliver NADPH or low potential one-electron donors (ferredoxin, flavodoxin) to redox-based metabolisms in plastids, mitochondria and bacteria. The plant-type reductase is also the basic prototype for one of the major families of flavin-containing electron transferases that display common functional and structural properties. Many aspects of FNR biochemistry have been extensively characterized in recent years using a combination of site-directed mutagenesis, steady-state and transient kinetic experiments, spectroscopy and X-ray crystallography. Despite these considerable advances, various key features in the enzymology of these important reductases remain yet to be explained in molecular terms. This article reviews the current status of these open questions. Measurements of electron transfer rates and binding equilibria indicate that NADP(H) and ferredoxin interactions with FNR result in a reciprocal decrease of affinity, and that this induced-fit step is a mandatory requisite for catalytic turnover. However, the expected conformational movements are not apparent in the reported atomic structures of these flavoenzymes in the free state or in complex with their substrates. The overall reaction catalysed by FNR is freely reversible, but the pathways leading to NADP+ or ferredoxin reduction proceed through entirely different kinetic mechanisms. Also, the reductases isolated from various sources undergo inactivating denaturation on exposure to NADPH and other electron donors that reduce the FAD prosthetic group, a phenomenon that might have profound consequences for FNR function in vivo. The mechanisms underlying this reductive inhibition are so far unknown. Finally, we provide here a rationale to interpret FNR evolution in terms of catalytic efficiency. Using the formalism of the Albery-Knowles theory, we identified which parameter(s) have to be modified to make these reductases even more proficient under a variety of conditions, natural or artificial. Flavoenzymes with FNR activity catalyse a number of reactions with potential importance for biotechnological processes, so that modification of their catalytic competence is relevant on both scientific and technical grounds.
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                Author and article information

                Contributors
                jgrzyb@ifpan.edu.pl
                mariusz.gagos@up.lublin.pl
                b.mysliwa-kurdziel@uj.edu.pl
                monika.bojko@uj.edu.pl
                wieslaw.gruszecki@umcs.pl
                andrzej.waloszek@uj.edu.pl
                strzalka@mol.uj.edu.pl
                Journal
                J Biol Phys
                J Biol Phys
                Journal of Biological Physics
                Springer Netherlands (Dordrecht )
                0092-0606
                1573-0689
                2 February 2012
                2 February 2012
                June 2012
                : 38
                : 3
                : 415-428
                Affiliations
                [1 ]Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Krakow, Poland
                [2 ]Laboratory of Biological Physics, Institute of Physics, PAS, al. Lotników 32/46, 02-668 Warsaw, Poland
                [3 ]Department of Biophysics, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland
                [4 ]Department of Cell Biology, Institute of Biology, Maria Curie-Skłodowska University, 20-033 Lublin, Poland
                [5 ]Department of Biophysics, Maria Sklodowska-Curie University, Lublin, Poland
                Article
                9262
                10.1007/s10867-012-9262-z
                3388194
                22912532
                e3918dab-9e10-4478-8681-5c7a52881518
                © The Author(s) 2012
                History
                : 26 August 2011
                : 3 January 2012
                Categories
                Original Paper
                Custom metadata
                © Springer Science+Business Media B.V. 2012

                Biophysics
                tertiary structure,secondary structure,ferredoxin:nadp+ oxidoreductase,heavy metals,cadmium

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