Abstract
[FeFe] hydrogenases are efficient catalysts for interconverting hydrogen with protons and electrons, whose catalytic mechanism remains a subject of controversy. Their active site, the H-cluster, is composed of a [4Fe–4S] subcluster ([4Fe–4S]H) covalently attached to a [2Fe] subcluster ([2Fe]H). The two subclusters are strongly redox-coupled, and proton-coupled electron transfer (PCET) within the H-cluster is thought to be essential for catalytic activity. Additionally, proton-coupled reduction of [4Fe–4S]H has been proposed. Here, we investigated the pH dependence of the redox behavior of [4Fe–4S]H using infrared (IR) spectroelectrochemistry with two different [FeFe] hydrogenases: CrHydA1 from Chlamydomonas reinhardtii and CpHydA1 from Clostridium pasteurianum. Contrary to previous reports, we find that, under our experimental conditions, the redox potential of [4Fe–4S]H is independent of pH around physiological values for both enzymes. We also found redox anticooperativity behavior between [4Fe–4S]H and the accessory [4Fe–4S] clusters (F-clusters) in CpHydA1, which tunes catalysis at the active site. Taken together, these results indicate that the catalytic cycle of [FeFe] hydrogenases likely does not involve protonation at or near [4Fe–4S]H, and instead, favors a model in which protonation of [2Fe]H drives catalysis. Our findings shed new light on the catalytic mechanism of [FeFe] hydrogenases and highlight the importance of the F-clusters in fine-tuning catalysis.
Highlights
ABSTRACT: [FeFe] hydrogenases are efficient catalysts for interconverting hydrogen with protons and electrons, whose catalytic mechanism remains a subject of controversy
Under our experimental conditions, the redox potential of [4Fe−4S]H is pH-independent in both enzymes studied, supporting Model 1 of the catalytic cycle in which protonation occurs at the ADT ligand, facilitating a protoncoupled electronic rearrangement within the H-cluster
As we have demonstrated that the redox potential of [4Fe− 4S]H does not depend on pH neither in CrHydA1 nor in CpHydA1, we believe that the redox potential of
Summary
ABSTRACT: [FeFe] hydrogenases are efficient catalysts for interconverting hydrogen with protons and electrons, whose catalytic mechanism remains a subject of controversy Their active site, the H‐cluster, is composed of a [4Fe−4S] subcluster ([4Fe−4S]H) covalently attached to a [2Fe] subcluster ([2Fe]H). In the first model (Figure 1D), oneelectron reduction of the enzyme in the Hox state gives a mixture of two different 1 e− reduced states (Hred and HredH+), one in which the electron resides mostly on [4Fe−4S]H and [2Fe]H remains mixed-valent (Hred), and the other in which the electron resides mostly on [2Fe]H, yielding a reduced Fe(I)Fe(I) subcluster and oxidized [4Fe−4S]H (HredH+).[13] These two reduced states were coined the Hred and HredH+ states because their populations varied in a pH-dependent fashion, with an apparent pKa of ≈7.2.12 At low pH, the electron remains mostly on [2Fe]H, and so it was assumed that protonation of the nitrogen in the bridging ADT ligand facilitated electronic rearrangement of the H-cluster to move the electron from [4Fe−4S]H (Hred) to [2Fe]H, leading to HredH+
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