Presenting Author The Pennsylvania State University
[FeFe] hydrogenases are enzymes that catalyze the reversible reduction of protons to dihydrogen with staggering efficiency. As dihydrogen is a promising alternative energy carrier for renewable energy systems, these enzymatic systems are of interest to biotechnological applications. Members of the [FeFe] hydrogenase class are very diverse in the primary structure, especially in the metallocofactor composition, and their physiological roles. Understanding the structure-function relationships in these enzymes provides an opportunity to engineer enzymes with uniquely modified characteristics. In this work, we aim to understand the interplay between structural elements and the phenotype of an unusual metalloprotein from Clostridium perfringens (CpHydR) containing two catalytic domains, [FeFe] hydrogenase and rubrerythrin, "fused" together into one protein. Although [FeFe] hydrogenases are typically biased toward H2 production, the postulated presence of rubrerythrin makes us hypothesize that CpHydR is biased toward H2 oxidation. Rubrerythrin is known to reduce H2O2 as a part of an oxidative stress response process; it requires the donation of two electrons and two protons, which can be supplied by the [FeFe] hydrogenase. Hydrogenases are notoriously oxygen-sensitive enzymes inhibiting their industrial application. The postulated use of this metalloprotein in the oxidative stress response in Clostridium perfringens would suggest tolerance to O2. Here we present experiments confirming the presence of each subsystem and the postulated functionality. We utilize Fourier Transformed Infrared (FTIR) and Electron Paramagnetic Resonance (EPR) spectroscopies to elucidate the structure of the active cofactors. To understand the function of CpHydR, we employ electrochemical methods, including protein film voltammetry. Results indicate an intriguing deviation of the spectroscopic signatures of the [FeFe] hydrogenase subdomain from the commonly observed phenotypes of virtually any other known enzyme of this class. This divergence in the structural characteristics also coincides with an unexpected strong bias of this enzyme towards hydrogen oxidation.
