Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase.

Area of Science:

• Biochemistry
• Enzyme kinetics
• Bioenergetics

Background:

• Redox enzymes like hydrogenases feature buried active sites linked to the solvent via molecular channels.
• The structure of these channels is hypothesized to influence enzyme selectivity and substrate/inhibitor interactions.
• Kinetic data on the functional role of these channels remain limited, despite insights from crystallography and molecular dynamics.

Purpose of the Study:

• To investigate the role of molecular channels in controlling substrate and inhibitor access to the active sites of NiFe and FeFe hydrogenases.
• To quantitatively correlate channel properties with enzyme kinetics, specifically diffusion rates and inhibition by CO and O2.

Main Methods:

• Utilized protein film voltammetry to measure kinetic parameters.
• Compared rates of inhibition by carbon monoxide (CO) and oxygen (O2) in ten NiFe hydrogenase mutants and two FeFe hydrogenases.
• Systematically modified amino acids within the enzyme's molecular channel.

Main Results:

• The rate of CO inhibition serves as a reliable indicator for O2 diffusion to the active site.
• Alterations to amino acid residues lining the channel significantly reduced O2 diffusion rates, by orders of magnitude.
• Established quantitative relationships between substrate diffusion, Michaelis constant for H2, and inhibition rates.

Conclusions:

• Enzyme active site channel structure is a critical determinant of substrate and inhibitor diffusion rates.
• Slow O2 inactivation in certain hydrogenases is attributed to restricted access to the active site through their molecular channels.
• Protein film voltammetry provides a powerful tool for dissecting the kinetic contributions of enzyme internal pathways.