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>

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>

Site-selective Protonation of the Catalytic Cofactor in [FeFe]-Hydrogenase

Hydrogenases are microbial redox enzymes that catalyze H2 oxidation and proton reduction (H2 evolution). While all hydrogenases show high H2 oxidation activities, [FeFe]-hydrogenases are excellent H2 evolution catalysts as well. Their hydrogen-forming active site cofactor (“H-cluster”) comprises a [4Fe-4S] cluster covalently linked to a diiron site modified with CO and CN– ligands that tune the redox potential and anchor the cofactor within the protein. Two distinct proton transfer pathways connect the H-cluster and facilitate hydrogen turnover at low overpotential. In this study, we employ in situ ATR FTIR spectroscopy and spectro-electrochemistry to analyze the mechanics of hydrogen turnover in [FeFe]-hydrogenase. Titrating the proton concentration from pH 10 to pH 5 under H2 oxidation or H2 evolution conditions reveals the influence of site-selective protonation on the equilibrium of H-cluster redox states. Under H2 evolution conditions, protonation facilitates electron uptake at the [4Fe-4S] cluster and impedes premature reduction of the diiron site, the later which dominates at acidic pH values. This observation is discussed in the context of the natural pH dependence of H2 evolution as catalyzed by [FeFe]-hydrogenase.<br>

Site-selective Protonation Controls Hydrogen Turnover in [FeFe]-Hydrogenase

Hydrogenases are metalloenzymes that catalyze H2 oxidation and H2 evolution. In particular, [FeFe]-hydrogenases have been shown to be excellent H2 evolution catalysts. Their active site cofactor comprises a [4Fe-4S] cluster covalently linked to a diiron site modified with CO and CN– ligands (H-cluster). Distinct proton transfer pathways connect the H-cluster on opposing sites facilitating hydrogen turnover at low overptential. In this study, we employ in situ ATR FTIR spectroscopy and spectro-electrochemistry to analyze H2 oxidation and H2 evolution of [FeFe]-hydrogenase at a wide range of proton concentrations. Our data reveals the influence of site-selective proton transfer on the equilibrium of redox states. Under reducing conditions, protonation facilitates electron uptake at the [4Fe-4S] cluster and impedes premature reduction of the diiron site, the later which dominates at acidic pH values. This observation is discussed in the context of the pH dependence of H2 evolution as catalyzed by [FeFe]-hydrogenase.<br>

Reaction of O2with a diiron protein generates a mixed-valent Fe2+/Fe3+center and peroxide

The gene encoding the cyanobacterial ferritinSynFtn is up-regulated in response to copper stress. Here, we show that, whileSynFtn does not interact directly with copper, it is highly unusual in several ways. First, its catalytic diiron ferroxidase center is unlike those of all other characterized prokaryotic ferritins and instead resembles an animal H-chain ferritin center. Second, as demonstrated by kinetic, spectroscopic, and high-resolution X-ray crystallographic data, reaction of O2with the di-Fe2+center results in a direct, one-electron oxidation to a mixed-valent Fe2+/Fe3+form. Iron–O2chemistry of this type is currently unknown among the growing family of proteins that bind a diiron site within a four α-helical bundle in general and ferritins in particular. The mixed-valent form, which slowly oxidized to the more usual di-Fe3+form, is an intermediate that is continually generated during mineralization. Peroxide, rather than superoxide, is shown to be the product of O2reduction, implying that ferroxidase centers function in pairs via long-range electron transfer through the protein resulting in reduction of O2bound at only one of the centers. We show that electron transfer is mediated by the transient formation of a radical on Tyr40, which lies ∼4 Å from the diiron center. As well as demonstrating an expansion of the iron–O2chemistry known to occur in nature, these data are also highly relevant to the question of whether all ferritins mineralize iron via a common mechanism, providing unequivocal proof that they do not.