Shedding Light on Proton and Electron Dynamics in [FeFe] Hydrogenases

Abstract

for reversible dihydrogen evolution. H2 turnover involves different catalytic intermediates including a recently characterized hydride state of the active site (H-cluster). Applying cryogenic infrared and electron paramagnetic resonance spectroscopy to an [FeFe] model hydrogenase from Chlamydomonas reinhardtii (CrHydA1), we have discovered two new hydride intermediates and spectroscopic evidence for a bridging CO ligand in two reduced H-cluster states.Our study provides novel insights into these key intermediates, their relevance for the catalytic cycle of [FeFe] hydrogenase, and novel strategies for exploring these aspects in detail.

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

“…The existence of alternative hydride-like states have been suggested earlier from work on HydA1, oen in amino acid variants with impaired proton transfer capacity, but has so far been challenging to accumulate and consequently not characterized in detail. [32][33][34][35]37,40 Herein, we show how such an alternative hydride-like state, H hyd H + , can be selectively generated also in the fully functional enzyme, not only in vitro but also under whole-cell conditions. The assignment of H hyd H + as a hydride state is conrmed by the observed H/D isotopic shi of the m-CO ligand.…”

“…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.…”