Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase.
In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a molecular channel whose structure may determine the enzyme's selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallography and molecular dynamics, but kinetic data are scarce. Using protein film voltammetry, we determined and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quantitatively define the relations between diffusion, the Michaelis constant for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow.
Liebgott
Pierre-Pol
PP
Centre National de la Recherche Scientifique, Unité Propre de Recherche 9036, Unité de Bioénergétique et Ingénierie des Protéines, Institut Fédératif de Recherche 88, Institut de Microbiologie de la Méditerranée, Marseille, France.
Leroux
Fanny
F
Burlat
Bénédicte
B
Dementin
Sébastien
S
Baffert
Carole
C
Lautier
Thomas
T
Fourmond
Vincent
V
Ceccaldi
Pierre
P
Cavazza
Christine
C
Meynial-Salles
Isabelle
I
Soucaille
Philippe
P
Fontecilla-Camps
Juan Carlos
JC
Guigliarelli
Bruno
B
Bertrand
Patrick
P
Rousset
Marc
M
Léger
Christophe
C
D000596:Amino Acids / Q000737:chemistry; D002248:Carbon Monoxide / Q000737:chemistry; D020134:Catalytic Domain; D018360:Crystallography, X-Ray / Q000379:methods; D003901:Desulfovibrio / Q000201:enzymology*; D004058:Diffusion; D004563:Electrochemistry / Q000379:methods; D004578:Electron Spin Resonance Spectroscopy; D006859:Hydrogen / Q000737:chemistry; D006864:Hydrogenase / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D010100:Oxygen / Q000737:chemistry*
D000596:Amino Acids; D002248:Carbon Monoxide; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen