The iron (Fe) proteins of molybdenum (Mo) and vanadium (V) nitrogenases mimic carbon monoxide (CO) dehydrogenase in catalyzing the interconversion between CO and CO under ambient conditions. Catalytic reduction of CO to CO is achieved in vitro and in vivo upon redox changes of the Fe-protein-associated [FeS] clusters. These observations establish the Fe protein as a model for investigation of CO activation while suggesting its biotechnological adaptability for recycling the greenhouse gas into useful products.
Hydrogenases comprise a variety of enzymes that catalyze the reversible oxidation of molecular hydrogen. Out of this group, [FeFe]-hydrogenase shows the highest activity for hydrogen production which is, therefore, of great interest in the field of renewable energies. Unfortunately, this comes with the flaw of a generally very high sensitivity against molecular oxygen that irreversibly inhibits this enzyme. While many studies have already addressed the mechanism of hydrogen formation by [FeFe]-hydrogenase, little is known about the molecular and mechanistic details leading to enzyme inactivation by O(2). In order to elucidate this process, we performed density functional theory calculations on several possible O(2) adducts of the catalytic center--the so-called H-cluster--and show that the direct interaction of the [2Fe](H) subsite with dioxygen is an exothermic and specific reaction in which O(2) most favorably binds in an end-on manner to the distal Fe(d). Based on the results, we propose a protonation mechanism that can explain the irreversibility of dioxygen-induced enzyme inactivation by water release and degradation of the ligand environment of the H-cluster.
NifB is an essential radical S-adenosylmethionine (SAM) enzyme for nitrogenase cofactor assembly. Previous studies show that NifB couples a putative pair of [Fe4S4] modules (designated K1 and K2) into an [Fe8S9C] cofactor precursor concomitant with radical SAM-dependent carbide insertion through the action of its SAM-binding [Fe4S4] module. However, the coordination and function of the NifB cluster modules remain unknown. Here, we use continuous wave and pulse electron paramagnetic resonance spectroscopy to show that K1- and K2-modules are 3-cysteine-coordinated [Fe4S4] clusters, with a histidine-derived nitrogen serving as the fourth ligand to K1 that is lost upon K1/K2-coupling. Further, we demonstrate that coexistence of SAM/K2-modules is a prerequisite for methyltransfer to K2 and hydrogen abstraction from the K2-associated methyl by a 5′-deoxyadenosyl radical. These results establish an important framework for mechanistic explorations of NifB while highlighting the utility of a synthetic-cluster-based reconstitution approach employed herein in functional analyses of iron–sulfur (FeS) enzymes.
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