How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms

Green algae such as Chlamydomonas reinhardtii synthesize an [FeFe] hydrogenase that is highly active in hydrogen evolution. However, the extreme sensitivity of [FeFe] hydrogenases to oxygen presents a major challenge for exploiting these organisms to achieve sustainable photosynthetic hydrogen production. In this study, the mechanism of oxygen inactivation of the [FeFe] hydrogenase CrHydA1 from C. reinhardtii has been investigated. X-ray absorption spectroscopy shows that reaction with oxygen results in destruction of the [4Fe-4S] domain of the active site H-cluster while leaving the di-iron domain (2FeH) essentially intact. By protein film electrochemistry we were able to determine the order of events leading up to this destruction. Carbon monoxide, a competitive inhibitor of CrHydA1 which binds to an Fe atom of the 2FeH domain and is otherwise not known to attack FeS clusters in proteins, reacts nearly two orders of magnitude faster than oxygen and protects the enzyme against oxygen damage. These results therefore show that destruction of the [4Fe-4S] cluster is initiated by binding and reduction of oxygen at the di-iron domain—a key step that is blocked by carbon monoxide. The relatively slow attack by oxygen compared to carbon monoxide suggests that a very high level of discrimination can be achieved by subtle factors such as electronic effects (specific orbital overlap requirements) and steric constraints at the active site.

[1]  F. Armstrong,et al.  The difference a Se makes? Oxygen-tolerant hydrogen production by the [NiFeSe]-hydrogenase from Desulfomicrobium baculatum. , 2008, Journal of the American Chemical Society.

[2]  M. Haumann,et al.  The structure of the active site H-cluster of [FeFe] hydrogenase from the green alga Chlamydomonas reinhardtii studied by X-ray absorption spectroscopy. , 2009, Biochemistry.

[3]  J. W. Peters,et al.  Dithiomethylether as a ligand in the hydrogenase h-cluster. , 2008, Journal of the American Chemical Society.

[4]  Kevin R. Messner,et al.  Mechanism of Superoxide and Hydrogen Peroxide Formation by Fumarate Reductase, Succinate Dehydrogenase, and Aspartate Oxidase* , 2002, The Journal of Biological Chemistry.

[5]  F. Armstrong,et al.  Electrochemical investigations of the interconversions between catalytic and inhibited states of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans. , 2006, Journal of the American Chemical Society.

[6]  P. Vignais,et al.  Occurrence, classification, and biological function of hydrogenases: an overview. , 2007, Chemical reviews.

[7]  J. Fontecilla-Camps,et al.  Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual coordination to an active site Fe binuclear center. , 1999, Structure.

[8]  Xin Yang,et al.  Synthesis of the H-cluster framework of iron-only hydrogenase , 2005, Nature.

[9]  S. Ott,et al.  Facilitated hydride binding in an Fe-Fe hydrogenase active-site biomimic revealed by X-ray absorption spectroscopy and DFT calculations. , 2007, Inorganic chemistry.

[10]  F. Armstrong,et al.  Investigations of the oxidative disassembly of Fe-S clusters in Clostridium pasteurianum 8Fe ferredoxin using pulsed-protein-film voltammetry. , 2000, Biochemistry.

[11]  Yue-qin Tang,et al.  Contrasting Sensitivities of Escherichia coli Aconitases A and B to Oxidation and Iron Depletion , 2003, Journal of bacteriology.

[12]  W. Lubitz,et al.  The electronic structure of the H-cluster in the [FeFe]-hydrogenase from Desulfovibrio desulfuricans: a Q-band 57Fe-ENDOR and HYSCORE study. , 2007, Journal of the American Chemical Society.

[13]  B J Lemon,et al.  X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution. , 1998, Science.

[14]  F. Armstrong,et al.  Investigating and exploiting the electrocatalytic properties of hydrogenases. , 2007, Chemical reviews.

[15]  M. Ghirardi,et al.  [FeFe]-hydrogenase-catalyzed H2 production in a photoelectrochemical biofuel cell. , 2008, Journal of the American Chemical Society.

[16]  W. Lubitz,et al.  [NiFe] and [FeFe] hydrogenases studied by advanced magnetic resonance techniques. , 2007, Chemical reviews.

[17]  L. K. Andersen,et al.  Reduction of Unusual Iron-Sulfur Clusters in the H2-sensing Regulatory Ni-Fe Hydrogenase from Ralstonia eutropha H16* , 2005, Journal of Biological Chemistry.

[18]  G. Kubas,et al.  Fundamentals of H2 binding and reactivity on transition metals underlying hydrogenase function and H2 production and storage. , 2007, Chemical reviews.

[19]  M. Gibbs,et al.  Inactivation of Hydrogenase in Cell-free Extracts and Whole Cells of Chlamydomonas reinhardi by Oxygen. , 1979, Plant physiology.

[20]  B. J. Lemon,et al.  Binding of exogenously added carbon monoxide at the active site of the iron-only hydrogenase (CpI) from Clostridium pasteurianum. , 1999, Biochemistry.

[21]  Jason C. Crack,et al.  Superoxide-mediated amplification of the oxygen-induced switch from [4Fe-4S] to [2Fe-2S] clusters in the transcriptional regulator FNR , 2007, Proceedings of the National Academy of Sciences.

[22]  A. L. Lacey,et al.  The active site of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans. II. Redox properties, light sensitivity and CO-ligand exchange as observed by infrared spectroscopy , 2005, JBIC Journal of Biological Inorganic Chemistry.

[23]  T. Happe,et al.  Optimized over-expression of [FeFe] hydrogenases with high specific activity in Clostridium acetobutylicum , 2008 .

[24]  M. Adams,et al.  The structure and mechanism of iron-hydrogenases. , 1990, Biochimica et biophysica acta.

[25]  Yvain Nicolet,et al.  Structure/function relationships of [NiFe]- and [FeFe]-hydrogenases. , 2007, Chemical reviews.

[26]  Robert K. Poole,et al.  Globins and other nitric oxide-reactive proteins , 2008 .

[27]  J. Imlay.  Iron‐sulphur clusters and the problem with oxygen , 2006, Molecular microbiology.

[28]  T. Happe,et al.  Isolation and molecular characterization of the [Fe]-hydrogenase from the unicellular green alga Chlorella fusca. , 2002, Biochimica et biophysica acta.

[29]  B. Guigliarelli,et al.  Hydrogen-activating enzymes: activity does not correlate with oxygen sensitivity. , 2008, Angewandte Chemie.

[30]  Erwin Reisner,et al.  Dynamic electrochemical investigations of hydrogen oxidation and production by enzymes and implications for future technology. , 2009, Chemical Society reviews.

[31]  V. Fernández,et al.  Crystallographic and FTIR spectroscopic evidence of changes in Fe coordination upon reduction of the active site of the Fe-only hydrogenase from Desulfovibrio desulfuricans. , 2001, Journal of the American Chemical Society.

[32]  Holger Dau,et al.  X-ray absorption spectroscopy to analyze nuclear geometry and electronic structure of biological metal centers—potential and questions examined with special focus on the tetra-nuclear manganese complex of oxygenic photosynthesis , 2003, Analytical and bioanalytical chemistry.

[33]  F. Armstrong,et al.  Influence of electrochemical properties in determining the sensitivity of [4Fe-4S] clusters in proteins to oxidative damage. , 2001, The Biochemical journal.

[34]  M. Ghirardi,et al.  Photobiological hydrogen-producing systems. , 2009, Chemical Society reviews.

[35]  A. L. Lacey,et al.  FTIR Characterization of the Active Site of the Fe-hydrogenase from Desulfovibrio desulfuricans , 2000 .

[36]  B. G. Searle,et al.  New Tools for the Analysis of EXAFS: The DL EXCURV Package , 2005 .

[37]  F. Armstrong,et al.  Hydrogen production under aerobic conditions by membrane-bound hydrogenases from Ralstonia species. , 2008, Journal of the American Chemical Society.

[38]  F. Albert Cotton,et al.  Advanced Inorganic Chemistry , 1999 .

[39]  W. Lubitz,et al.  Isolation and first EPR characterization of the [FeFe]-hydrogenases from green algae. , 2008, Biochimica et biophysica acta.

[40]  Robert Robson,et al.  Hydrogen as a Fuel : Learning from Nature , 2001 .