Inhibition and aerobic inactivation kinetics of Desulfovibrio fructosovorans NiFe hydrogenase studied by protein film voltammetry.
We have used protein film voltammetry to study the NiFe hydrogenase from Desulfovibrio fructosovorans. We show how measurements of transient activity following the addition in the electrochemical cell of H(2), CO, or O(2) allow simple and virtually instantaneous determinations of the Michaelis constant, inhibition constant, or rate of inactivation, respectively, thus opening new opportunities to study the active site of NiFe hydrogenases. The binding and release of CO occur within a fraction of a second, and we determine and discuss how its affinity for the active site changes as the driving force for the H(+)/H(2) reaction is continuously varied. Inactivation by O(2) is a slow, bimolecular process (with pH-independent rate constant approximately 3 x 10(4) s(-1) M(-1) at 40 degrees C, under one atm of H(2)) that leads to a mixture of fully oxidized states, and unlike the case of CO inhibition, the active site is not fully protected by H(2). This experimental approach could be used to study the reaction of other multicentered metalloenzymes with their gaseous substrates or inhibitors.
Léger
Christophe
C
Unité de Bioénergétique et Ingénierie des Protéines, Institut de Biologie Structurale et Microbiologie, CNRS UPR9036 et Université de Provence, 31, chemin Joseph Aiguier, 13402, Marseille, France. christophe.leger@ibsm.cnrs-mrs.fr
Dementin
Sébastien
S
Bertrand
Patrick
P
Rousset
Marc
M
Guigliarelli
Bruno
B
D000332:Aerobiosis; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D002248:Carbon Monoxide / Q000737:chemistry; D002841:Chromatiaceae / Q000201:enzymology; D003901:Desulfovibrio / Q000201:enzymology*; D004563:Electrochemistry / Q000379:methods; D004789:Enzyme Activation; D006859:Hydrogen / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D011312:Pressure
D001426:Bacterial Proteins; D002248:Carbon Monoxide; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen