The active site of [FeFe] hydrogenases, the H-cluster, consists of a [4Fe-4S] cluster connected via a bridging cysteine to a [2Fe] complex carrying CO and CN ligands as well as a bridging aza-dithiolate ligand (ADT) of which the amine moiety serves as a proton shuttle between the protein and the H-cluster. During the catalytic cycle, the two subclusters change oxidation states: [4Fe-4S] ⇔ [4Fe-4S] and [Fe(I)Fe(II)] ⇔ [Fe(I)Fe(I)] thereby enabling the storage of the two electrons needed for the catalyzed reaction 2H + 2e ⇄ H. Using FTIR spectro-electrochemistry on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) at different pH values, we resolve the redox and protonation events in the catalytic cycle and determine their intrinsic thermodynamic parameters. We show that the singly reduced state H of the H-cluster actually consists of two species: H = [4Fe-4S] - [Fe(I)Fe(II)] and HH = [4Fe-4S] - [Fe(I)Fe(I)] (H) related by proton coupled electronic rearrangement. The two redox events in the catalytic cycle occur on the [4Fe-4S] subcluster at similar midpoint-potentials (-375 vs -418 mV); the protonation event (H/HH) has a pK ≈ 7.2.
Sensory type [FeFe] hydrogenases are predicted to play a role in transcriptional regulation by detecting the H level of the cellular environment. These hydrogenases contain the hydrogenase domain with distinct modifications in the active site pocket, followed by a Per-Arnt-Sim (PAS) domain. As yet, neither the physiological function nor the biochemical or spectroscopic properties of these enzymes have been explored. Here, we present the characterization of an artificially maturated, putative sensory [FeFe] hydrogenase from Thermotoga maritima (HydS). This enzyme shows lower hydrogen conversion activity than prototypical [FeFe] hydrogenases and a reduced inhibition by CO. Using FTIR spectroelectrochemistry and EPR spectroscopy, three redox states of the active site were identified. The spectroscopic signatures of the most oxidized state closely resemble those of the H state from the prototypical [FeFe] hydrogenases, while the FTIR spectra of both singly and doubly reduced states show large differences. The FTIR bands of both the reduced states are strongly red-shifted relative to the H state, indicating reduction at the diiron site, but with retention of the bridging CO ligand. The unique functional and spectroscopic features of HydS are discussed with regard to the possible role of altered amino acid residues influencing the electronic properties of the H-cluster.
[FeFe] hydrogenases catalyze proton reduction and hydrogen oxidation displaying high rates at low overpotential. Their active site is a complex cofactor consisting of a unique [2Fe] subcluster ([2Fe]) covalently bound to a canonical [4Fe-4S] cluster ([4Fe-4S]). The [FeFe] hydrogenase from Desulfovibrio desulfuricans is exceptionally active and bidirectional. This enzyme features two accessory [4Fe-4S] clusters for exchanging electrons with the protein surface. A thorough understanding of the mechanism of this efficient enzyme will facilitate the development of synthetic molecular catalysts for hydrogen conversion. Here, it is demonstrated that the accessory clusters influence the catalytic properties of the enzyme through a strong redox interaction between the proximal [4Fe-4S] cluster and the [4Fe-4S] subcluster of the H-cluster. This interaction enhances proton-coupled electronic rearrangement within the H-cluster increasing the apparent pK of its one electron reduced state. This may help to sustain H production at high pH values. These results may apply to all [FeFe] hydrogenases containing accessory clusters.
Background: Light-harvesting complex II (LHCII) displays different spectroscopic properties in thylakoid membranes and in detergents. Results: Circular dichroism and fluorescence lifetimes disclose structural effects of protein-protein, lipid-protein, and detergent interactions.
Conclusion:The native state of complexes is perturbed by detergents and best retained in lipid:LHCII assemblies. Significance: The lipid environment is important for the proper function of LHCII, the most abundant of membrane proteins.
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