EPR and electron nuclear double resonance investigation of oxidized hydrogenase II (uptake) from Clostridium pasteurianum W5. Effects of carbon monoxide binding.
Two different hydrogenases have been isolated from Clostridium pasteurianum W5. Hydrogenase II (uptake) is active in H2 oxidation while hydrogenase I (bidirectional) is active both in H2 oxidation and evolution. Previous EPR and electron nuclear double resonance (ENDOR) studies of oxidized hydrogenase I have now been complemented by analogous studies on oxidized 57Fe-enriched hydrogenase II and its CO derivative (using 12CO and 13CO). Binding of CO greatly changes the EPR spectrum of oxidized hydrogenase II, and use of 13CO leads to resolved hyperfine splitting from interaction with a single 13CO molecule (AC approximately 34 MHz). This coupling is over 50% larger than that seen for hydrogenase I. 57Fe ENDOR disclosed two types of iron site in both oxidized hydrogenase II and its CO derivative. Combination of EPR, ENDOR, and Mössbauer results shows that site 1 has AFe1 = 18 MHz shifting to approximately 30 MHz upon CO binding and consisting of two Fe atoms and site 2 has A2 approximately 7 MHz shifting to approximately 10 MHz and containing a single Fe. These results are very similar to those seen for hydrogenase I, which indicates that a structurally similar 3Fe cluster, believed to be the catalytically active site, is present in both. Proton ENDOR shows a solvent exchangeable resonance only in the CO derivative of hydrogenase II. This indicates a structural difference between hydrogenases I and II that is brought out by CO binding. No evidence of 14N coordination to the cluster is seen for either enzyme.
D002248:Carbon Monoxide / Q000494:pharmacology*; D003013:Clostridium / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D011485:Protein Binding
D002248:Carbon Monoxide; D010088:Oxidoreductases; C029746:uptake hydrogenase