Proton-coupled electron transfer (PCET) is a fundamental process at the core of oxidation-reduction reactions for energy conversion in biology. The [FeFe]-hydrogenases catalyze reversible H2 activation through a unique metallocofactor, the H-cluster, that is finely tuned by the surrounding protein environment to undergo fast PCET transitions. The H-cluster is comprised of a [4Fe-4S]H cubane subcluster linked by cysteine (Cys) to an organometallic [2Fe]H subcluster with CO and CN- ligands. Defining the PCET steps that coordinate redox transitions at the H-cluster have been difficult to resolve experimentally due to inherently fast (≥104 s-1) enzyme turnover. We have recently investigated the effects of a Cys→Ser substitution at a conserved site (169) in the PT network of Chlamydomonas reinhardtii [FeFe]-hydrogenase (CrHydA1). Cys169 lies proximal to, and mediates proton exchange with, the H-cluster. The Cys169Ser variant of CrHydA1 showed a substantial decrease in catalytic activity, and the electron paramagnetic resonance (EPR) and Fourier transform infrared (FTIR) spectroscopic properties revealed enrichment of unique H-cluster states under reducing conditions. Reduction with H2 or sodium dithionite led to an EPR spectra with signals consistent with a reduced [4Fe-4S]H1+ subcluster, and FTIR spectra that showed upshifts of vCO modes to energies consistent with an increase in oxidation state of the [2Fe]H subcluster. Density Functional Theory analysis corroborated this redox transition and provided additional insights on shifts in [2Fe]H vCO modes during H2 activation. In contrast to wild-type CrHydA1, spectra associated with the so-called "reduced" states (Hred and Hsred) were less populated in the Cys→Ser variant, demonstrating that the exchange of -SH to -OH alters how the H-cluster equilibrates among reduced states of the catalytic cycle under steady-state conditions. This presentation will summarize the results in the context of a model for H2 activation by [FeFe]-hydrogenase and the role of proton-transfer in the catalytic cycle.

Spectroscopic studies of proton-coupled electron transfer in the mechanism of H2 activation by [FeFe]-hydrogenases

249th National Meeting (2015)

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