Electrochemical Measurements of the Kinetics of Inhibition of Two FeFe Hydrogenases by O2 Demonstrate That the Reaction Is Partly Reversible.
The mechanism of reaction of FeFe hydrogenases with oxygen has been debated. It is complex, apparently very dependent on the details of the protein structure, and difficult to study using conventional kinetic techniques. Here we build on our recent work on the anaerobic inactivation of the enzyme [Fourmond et al. Nat. Chem. 2014, 4, 336-342] to propose and apply a new method for studying this reaction. Using electrochemical measurements of the turnover rate of hydrogenase, we could resolve the first steps of the inhibition reaction and accurately determine their rates. We show that the two most studied FeFe hydrogenases, from Chlamydomonas reinhardtii and Clostridium acetobutylicum, react with O2 according to the same mechanism, despite the fact that the former is much more O2 sensitive than the latter. Unlike often assumed, both enzymes are reversibly inhibited by a short exposure to O2. This will have to be considered to elucidate the mechanism of inhibition, before any prediction can be made regarding which mutations will improve oxygen resistance. We hope that the approach described herein will prove useful in this respect.
Laboratoire de Bioénergétique et Ingénierie des Protéines, CNRS/Aix-Marseille Université, 13402 Marseille, France.
Christophe
C
Orain
CEA, Institut de Biologie et de Technologies de Saclay IBITECS, SB2SM, F-91191 Gif sur Yvette, France.|Institut de Biologie Intégrative de la Cellule I2BC, UMR 9198, CEA, CNRS, Université Paris Sud , F-91191 Gif sur Yvette, France.
Laure
L
Saujet
Université de Toulouse, INSA, UPS, INP, LISBP, INRA:UMR792,135 CNRS:UMR 5504, avenue de Rangueil, 31077 Toulouse, France.
Charles
C
Gauquelin
Université de Toulouse, INSA, UPS, INP, LISBP, INRA:UMR792,135 CNRS:UMR 5504, avenue de Rangueil, 31077 Toulouse, France.
Philippe
P
Soucaille
Université de Toulouse, INSA, UPS, INP, LISBP, INRA:UMR792,135 CNRS:UMR 5504, avenue de Rangueil, 31077 Toulouse, France.
Isabelle
I
Meynial-Salles
Laboratoire de Bioénergétique et Ingénierie des Protéines, CNRS/Aix-Marseille Université, 13402 Marseille, France.
Carole
C
Baffert
Laboratoire de Bioénergétique et Ingénierie des Protéines, CNRS/Aix-Marseille Université, 13402 Marseille, France.
Vincent
V
Fourmond
CEA, Institut de Biologie et de Technologies de Saclay IBITECS, SB2SM, F-91191 Gif sur Yvette, France.|Institut de Biologie Intégrative de la Cellule I2BC, UMR 9198, CEA, CNRS, Université Paris Sud , F-91191 Gif sur Yvette, France.
Hervé
H
Bottin
Laboratoire de Bioénergétique et Ingénierie des Protéines, CNRS/Aix-Marseille Université, 13402 Marseille, France.
Christophe
C
Léger
D020134:Catalytic Domain; D004563:Electrochemistry; D006864:Hydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D010100:Oxygen / Q000737:chemistry*
D007506:Iron-Sulfur Proteins; C118830:iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen