Characterization of CpI and CpII [FeFe]-hydrogenases reveals properties contributing to catalytic bias

Abstract

The need for food, fuel, and pharmaceuticals has been increasing at a growing rate as the world's population increases and lifestyles improve. All of these needs are highly energy dependent, and, to a significant degree, rely on an inefficient use of fossil fuels. In order to break free of this dependence, new understanding is required for how to efficiently generate the products humanity needs. Here, a model system of two closely related [FeFe]-hydrogenases, CpI and CpII, is employed in order to understand how biology is able to efficiently control the formation of reduced products, in order to further delineate the limits of control, and the extent to which biology may be co-opted for technological needs. CpI, one of nature's best catalysts for reducing protons to hydrogen gas, is compared to CpII, which functions catalytically to oxidize hydrogen to protons and electrons. Oxygen sensitivity, midpoint potentials, catalytic mechanisms, and catalytic bias are explored in-depth using electron paramagnetic resonance, Fourier Transform Infrared spectroscopy, and protein film voltammetry. CpI and CpII have been found to function under different metabolic conditions, and key amino acids influencing their distinct behavior have been identified. The conduit arrays of hydrogenases, which direct electrons to or from the active site, have been found to have distinct midpoint potentials in CpII compared to CpI, effectively reversing the favored electron flow through CpII in comparison to CpI. In order to probe the contributions of the protein framework on catalysis, analysis of site-specific amino acid substituted variants have been used to identify several determinants that affect the H-cluster environment, which contributes to the observed differences between CpI and CpII. This has resulted in a deeper understanding of the hydrogenase model system and the ability to directly influence catalytic bias. Thus, the work presented here represents key progress towards developing unidirectional catalysts, and demonstrates the possibility of targeted, rational design and implementation of unidirectional catalysts.