5 research outputs found
Protonation/reduction dynamics at the [4Fe–4S] cluster of the hydrogen-forming cofactor in [FeFe]-hydrogenases
The [FeFe]-hydrogenases of bacteria and algae are the most efficient hydrogen
conversion catalysts in nature. Their active-site cofactor (H-cluster)
comprises a [4Fe–4S] cluster linked to a unique diiron site that binds three
carbon monoxide (CO) and two cyanide (CN−) ligands. Understanding microbial
hydrogen conversion requires elucidation of the interplay of proton and
electron transfer events at the H-cluster. We performed real-time spectroscopy
on [FeFe]-hydrogenase protein films under controlled variation of atmospheric
gas composition, sample pH, and reductant concentration. Attenuated total
reflection Fourier-transform infrared spectroscopy was used to monitor shifts
of the CO/CN− vibrational bands in response to redox and protonation changes.
Three different [FeFe]-hydrogenases and several protein and cofactor variants
were compared, including element and isotopic exchange studies. A protonated
equivalent (HoxH) of the oxidized state (Hox) was found, which preferentially
accumulated at acidic pH and under reducing conditions. We show that the one-
electron reduced state Hred′ represents an intrinsically protonated species.
Interestingly, the formation of HoxH and Hred′ was independent of the
established proton pathway to the diiron site. Quantum chemical calculations
of the respective CO/CN− infrared band patterns favored a cysteine ligand of
the [4Fe–4S] cluster as the protonation site in HoxH and Hred′. We propose
that proton-coupled electron transfer facilitates reduction of the [4Fe–4S]
cluster and prevents premature formation of a hydride at the catalytic diiron
site. Our findings imply that protonation events both at the [4Fe–4S] cluster
and at the diiron site of the H-cluster are important in the hydrogen
conversion reaction of [FeFe]-hydrogenases
Site-selective protonation of the one-electron reduced cofactor in [FeFe]-hydrogenase
Hydrogenases are bidirectional redox enzymes that catalyze hydrogen turnover in archaea, bacteria, and algae. While all types of hydrogenase show H-2 oxidation activity, [FeFe]-hydrogenases are excellent H-2 evolution catalysts as well. Their active site cofactor comprises a [4Fe-4S] cluster covalently linked to a diiron site equipped with carbon monoxide and cyanide ligands. The active site niche is connected with the solvent by two distinct proton transfer pathways. To analyze the catalytic mechanism of [FeFe]-hydrogenase, we employ operando infrared spectroscopy and infrared spectro-electrochemistry. Titrating the pH under H-2 oxidation or H-2 evolution conditions reveals the influence of site-selective protonation on the equilibrium of reduced cofactor states. Governed by pK(a) differences across the active site niche and proton transfer pathways, we find that individual electrons are stabilized either at the [4Fe-4S] cluster (alkaline pH values) or at the diiron site (acidic pH values). This observation is discussed in the context of the complex interdependence of hydrogen turnover and bulk pH
Site-selective Protonation of the One-electron Reduced Cofactor in [FeFe]-Hydrogenase
Hydrogenases are microbial redox enzymes
that catalyze H2 oxidation and proton reduction (H2 evolution). While
all hydrogenases show high oxidation activities, the majority of
[FeFe]-hydrogenases are excellent H2 evolution catalysts as well. Their
active site cofactor comprises a [4Fe-4S] cluster covalently linked to a
diiron site equipped with carbon monoxide and cyanide ligands that
facilitate catalysis at low overpotential. Distinct proton transfer
pathways connect the active site niche with the solvent, resulting in a
non-trivial dependence of hydrogen turnover and bulk pH. To analyze the
catalytic mechanism of [FeFe]-hydrogenase, we employ in situ infrared
spectroscopy and infrared spectro-electrochemistry. Titrating the pH
under H2 oxidation or H2 evolution conditions reveals the influence of
site-selective protonation on the equilibrium of reduced cofactor
states. Governed by pKa differences across the active site niche and
proton transfer pathways, we find that individual electrons are
stabilized either at the [4Fe-4S] cluster (alkaline pH values) or at the
diiron site (acidic pH values). This observation is discussed in the
context of the natural pH dependence of hydrogen turnover as catalyzed
by [FeFe]-hydrogenase.<br /
Comprehensive structural, infrared spectroscopic and kinetic investigations of the roles of the active-site arginine in bidirectional hydrogen activation by the [NiFe]-hydrogenase 'Hyd-2' from Escherichia coli
The active site of [NiFe]-hydrogenases contains a strictly-conserved pendant arginine, the guanidine head group of which is suspended immediately above the Ni and Fe atoms. Replacement of this arginine (R479) in hydrogenase-2 from E. coli results in an enzyme that is isolated with a very tightly-bound diatomic ligand attached end-on to the Ni and stabilised by hydrogen bonding to the Nζ atom of the pendant lysine and one of the three additional water molecules located in the active site of the variant. The diatomic ligand is bound under oxidising conditions and is removed only after a prolonged period of reduction with H2 and reduced methyl viologen. Once freed of the diatomic ligand, the R479K variant catalyses both H2 oxidation and evolution but with greatly decreased rates compared to the native enzyme. Key kinetic characteristics are revealed by protein film electrochemistry: most importantly, a very low activation energy for H2 oxidation that is not linked to an increased H/D isotope effect. Native electrocatalytic reversibility is retained. The results show that the sluggish kinetics observed for the lysine variant arise most obviously because the advantage of a more favourable low-energy pathway is massively offset by an extremely unfavourable activation entropy. Extensive efforts to establish the identity of the diatomic ligand, the tight binding of which is an unexpected further consequence of replacing the pendant arginine, prove inconclusive