Insight into the Redox Behavior of the [4Fe–4S] Subcluster in [FeFe] Hydrogenases

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.

Electrochemical control of [FeFe]-hydrogenase single crystals reveals complex redox populations at the catalytic site

“…The counterintuitive formation of Hred at potentials lower or higher than HredH + , as a function of pH, is in marked contrast with previous studies on CrHydA1, 13 where they coexist at the same potentials, but it correlates extremely well with studies on DdH. 11,21 The fact that CpI features this behaviour is likely due to redox anti-cooperativity between the H-cluster and the proximal F-cluster (present in both CpI and DdH but absent in CrHydA1). Some of the IR signals discussed above have been proposed to be due to the incorporation of a degraded form of the synthetic precursor [2Fe] adt in DdH (namely 1988 and 1921 cm -1 ).…”

“…[17][18][19] A 1-electron reduction of the H-cluster in Hox yields the reduced state Hred that features a similar geometry with a [4Fe4S] 1+ -Fe(II)Fe(I) configuration. 13,20,21 An alternative nomenclature refers to this species as Hred'. 15,22 The protonated reduced state HredH + (Hred according to the alternative nomenclature) displays a [4Fe4S] 2+ -Fe(I)Fe(I) configuration and can be obtained either by a single proton binding to Hred (with simultaneous intra-cluster electron transfer from [4Fe4S] to Fe p ), or by proton-coupled electron transfer (PCET) from Hox.…”

“…30,31 Separate protonation events have been proposed to take place on the cubane sub-cluster of the catalytic centre, resulting in a duplicate set of alternatively protonated redox states which have been termed HoxH, HoxHCO, Hred'H and HhydH. 15,21,22,32 The H-cluster redox states and reactivity have been extensively investigated in model enzymes, particularly CrHydA1 (from the green alga Chlamydomonas reinhardtii), CpI (from the anaerobic nitrogen-fixing bacterium Clostridium pasteurianum) and DdH (from the sulfate-reducing bacterium Desulfovibrio desulfuricans). 2,3,33 CaI (from the solvent-producer Clostridium acetobutylicum) has a high degree of homology with CpI and has likewise been characterised.…”

Site-selective protonation of the one-electron reduced cofactor in [FeFe]-hydrogenase

“…Surprisingly, Rodríguez-Macia et al found the redox potential of the [4Fe-4S] cluster in CrHydA1 PDT to be independent from bulk pH. 48 The reasons for this discrepancy are unclear. In our hands, present and previous data 31,38 support PCET in the formation of Hred′.…”

“…24,47 The influence of bulk pH on Hox, Hred, and Hsred in the native [FeFe]-hydrogenase from Chlamydomonas reinhardtii (CrHydA1) 32,39 and Hred′ in cofactor variant CrHydA1 PDT was analysed before. 31,48 To understand the equilibrium of Hred′ and Hred in native CrHydA1, we now investigate the pH-dependent accumulation of both 1e − -reduced H-cluster states under turnover conditions. Making use of operando attenuated total reflection Fourier-transform infrared (ATR FTIR) spectroscopy and spectro-electrochemistry under H 2 oxidation or H 2 evolution conditions, we found consistent trends for an accumulation of Hred′ towards alkaline pH values whereas the accumulation of Hred increases towards acidic pH values.…”