C. Leger, S. Dementin, P. Bertrand, M. Rousset and B. Guigliarelli
J. Am. Chem. Soc. 126, 12162 (2004) doi:10.1021/ja046548d
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 H2, CO, or O2 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+/H2 reaction is continuously varied. Inactivation by O2 is a slow, bimolecular process (with pH-independent rate constant ≈ 3 × 104 s-1 M-1 at 40 °C, under one atm of H2) that leads to a mixture of fully oxidized states, and unlike the case of CO inhibition, the active site is not fully protected by H2. This experimental approach could be used to study the reaction of other multicentered metalloenzymes with their gaseous substrates or inhibitors.