RESUMO
We demonstrate that the insertion of the dinuclear active site of [FeFe] hydrogenase into the apo-enzyme can occur when the enzyme is embedded in a film of redox polymer, under conditions of mediated electron transfer. The maturation can be monitored by electrochemistry, and is as fast as under conditions of direct electron transfer. This new approach further clears the way to the implementation of hydrogenases in large scale real life processes.
Assuntos
Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Hidrogenase/química , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Membranas Artificiais , Polímeros/químicaRESUMO
[FeFe]-hydrogenases, HydAs, are unique biocatalysts for proton reduction to H2. However, they suffer from a number of drawbacks for biotechnological applications: size, number and diversity of metal cofactors, oxygen sensitivity. Here we show that HydA from Megasphaera elsdenii (MeHydA) displays significant resistance to O2. Furthermore, we produced a shorter version of this enzyme (MeH-HydA), lacking the N-terminal domain harboring the accessory FeS clusters. As shown by detailed spectroscopic and biochemical characterization, MeH-HydA displays the following interesting properties. First, a functional active site can be assembled in MeH-HydA in vitro, providing the enzyme with excellent hydrogenase activity. Second, the resistance of MeHydA to O2 is conserved in MeH-HydA. Third, MeH-HydA is more biased toward proton reduction than MeHydA, as the result of the truncation changing the rate limiting steps in catalysis. This work shows that it is possible to engineer HydA to generate an active hydrogenase that combines the resistance of the most resistant HydAs and the simplicity of algal HydAs, containing only the H-cluster.
Assuntos
Hidrogenase/metabolismo , Megasphaera elsdenii/enzimologia , Oxigênio/metabolismo , Engenharia de Proteínas , Biocatálise , Monóxido de Carbono/metabolismo , Domínio Catalítico , Hidrogenase/química , Hidrogenase/genética , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Megasphaera elsdenii/química , Megasphaera elsdenii/genética , Megasphaera elsdenii/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Engenharia de Proteínas/métodosRESUMO
[FeFe] hydrogenase (HydA) catalyzes interconversion between 2H+ and H2 at an active site composed of a [4Fe-4S] cluster linked to a 2Fe subcluster that harbors CO, CN- and azapropanedithiolate (adt2-) ligands. HydE, HydG and HydF are the maturases specifically involved in the biosynthesis of the 2Fe subcluster. Using ligands synthesized by HydE and HydG, HydF assembles a di-iron precursor of the 2Fe subcluster and transfers it to HydA for maturation. Here we report the first X-ray structure of HydF with its [4Fe-4S] cluster. The cluster is chelated by three cysteines and an exchangeable glutamate, which allows the binding of synthetic mimics of the 2Fe subcluster. [Fe2(adt)(CO)4(CN)2]2- is proposed to be the true di-iron precursor because, when bound to HydF, it matures HydA and displays features in Fourier transform infrared (FTIR) spectra that are similar to those of the native HydF active intermediate. A new route toward the generation of artificial hydrogenases, as combinations of HydF and such biomimetic complexes, is proposed on the basis of the observed hydrogenase activity of chemically modified HydF.
