CO-Bridged H-Cluster Intermediates in the Catalytic Mechanism of [FeFe]-Hydrogenase CaI

Abstract

The [FeFe]-hydrogenases ([FeFe] Hases) catalyze reversible H activation at the H-cluster, which is composed of a [4Fe-4S] subsite linked by a cysteine thiolate to a bridged, organometallic [2Fe-2S] ([2Fe]) subsite. Profoundly different geometric models of the H-cluster redox states that orchestrate the electron/proton transfer steps of H bond activation have been proposed. We have examined this question in the [FeFe] Hase I from Clostridium acetobutylicum (CaI) by Fourier-transform infrared (FTIR) spectroscopy with temperature annealing and H/D isotope exchange to identify the relevant redox states and define catalytic transitions. One-electron reduction of H led to formation of HH ([4Fe-4S]-Fe-Fe) and H' ([4Fe-4S]-Fe-Fe), with both states characterized by low frequency μ-CO IR modes consistent with a fully bridged [2Fe]. Similar μ-CO IR modes were also identified for HH of the [FeFe] Hase from Chlamydomonas reinhardtii (CrHydA1). The CaI proton-transfer variant C298S showed enrichment of an H/D isotope-sensitive μ-CO mode, a component of the hydride bound H-cluster IR signal, H. Equilibrating CaI with increasing amounts of NaDT, and probed at cryogenic temperatures, showed HH was converted to H. Over an increasing temperature range from 10 to 260 K catalytic turnover led to loss of H and appearance of H, consistent with enzymatic turnover and H formation. The results show for CaI that the μ-CO of [2Fe] remains bridging for all of the "H" states and that HH is on pathway to H and H evolution in the catalytic mechanism. These results provide a blueprint for designing small molecule catalytic analogs.

New Approaches to an Old Problem: Original Techniques in [FeFe]-hydrogenase Research

“…83 Interestingly, cryogenic IR spectroscopy indicated a bridging ligand for both Hred and Hsred. [122][123][124] This is in agreement with a recent study by Artz et al comparing cryogenic CpI crystal structures solved with both synchrotron radiation and the free electron laser (XFEL) light source at Stanford. 47 The authors were able to quantify the extent of photoreduction suggesting that in the presence of strong reductant up to 50% of CpI was found in a "more reduced conformation" featuring the H-cluster in a µCO geometry.…”

“…For DdH, CrHydA1, and CaI these bands were assigned to a µCO ligand in Hred (~1810 ±7 cm -1 ) and Hsred (~1790 ±12 cm -1 ). [122][123][124] The experimental variance is surprisingly high, given that the µCO band is usually well conserved among Group A [FeFe]hydrogenases (Table 1). More importantly, the µCO frequencies are in the same range as Hox and Hred´ (Figure 8), which hints at a Fe(II)-Fe(I) configuration rather than Fe(I)-Fe(I).…”

“…132 Today, ambient and cryogenic measurements show both the presence of an additional terminal CO ligand at room temperature 110,115,119,121 and the presence of a µCO ligand at cryogenic temperature. [122][123][124] Moreover, the geometry of Hred and Hsred under ambient conditions is hotly debated, with regards to protonation sites and whether the diiron site adopts an H2inhibited, µH geometry or an active-ready, Hox-like geometry with a µCO ligand. 115 In order to understand the interconversion of room temperature and cryogenic species of the H-cluster, it will be important to address the influence of rotational freedom and protein environment as well as proton transfer and proton-coupled electron transport in the accumulation and 'shaping' of redox states as a function of temperature.…”

Spectroscopic and biochemical insight into an electron-bifurcating [FeFe] hydrogenase

“…7a), which may indicate that in the H red H + state the bridging CO becomes terminal, as was proposed for the H red H + state in the [FeFe] hydrogenases from Chlamydomonas reinhardtii (CrHydA1) and Desulfovibrio desulfuricans (DdHydAB) [8,44,45]. However, in a recent study, it was suggested that the bridging CO can be retained in the H red H + state under certain conditions [46].…”

Spectroscopic investigations under whole-cell conditions provide new insight into the metal hydride chemistry of [FeFe]-hydrogenase

“…28 The exact nature of these intermediate states and their relevance to the catalytic mechanism is under debate. 10,27,[30][31][32][33] However, all recent models agree that a terminal hydride is formed during catalysis. One such intermediate, denoted H hyd , has been detected in vitro and shown to accumulate in amino acid variants with disrupted proton transfer networks, but also in the native enzyme under reducing conditions at low pH.…”