Dataset Information

Hydrogen activation by biomimetic [NiFe]-hydrogenase model containing protected cyanide cofactors.

ABSTRACT: Described are experiments demonstrating incorporation of cyanide cofactors and hydride substrate into [NiFe]-hydrogenase (H2ase) active site models. Complexes of the type (CO)2(CN)2Fe(pdt)Ni(dxpe) (dxpe = dppe, 1; dxpe = dcpe, 2) bind the Lewis acid B(C6F5)3 (BAr(F)3) to give the adducts (CO)2(CNBAr(F)3)2Fe(pdt)Ni(dxpe), (1(BAr(F)3)2, 2(BAr(F)3)2). Upon decarbonylation using amine oxides, these adducts react with H2 to give hydrido derivatives [(CO)(CNBAr(F)3)2Fe(H)(pdt)Ni(dxpe)](-) (dxpe = dppe, [H3(BAr(F)3)2](-); dxpe = dcpe, [H4(BAr(F)3)2](-)). Crystallographic analysis shows that Et4N[H3(BAr(F)3)2] generally resembles the active site of the enzyme in the reduced, hydride-containing states (Ni-C/R). The Fe-H···Ni center is unsymmetrical with r(Fe-H) = 1.51(3) Å and r(Ni-H) = 1.71(3) Å. Both crystallographic and (19)F NMR analyses show that the CNBAr(F)3(-) ligands occupy basal and apical sites. Unlike cationic Ni-Fe hydrides, [H3(BAr(F)3)2](-) and [H4(BAr(F)3)2](-) oxidize at mild potentials, near the Fc(+/0) couple. Electrochemical measurements indicate that in the presence of base, [H3(BAr(F)3)2](-) catalyzes the oxidation of H2. NMR evidence indicates dihydrogen bonding between these anionic hydrides and R3NH(+) salts, which is relevant to the mechanism of hydrogenogenesis. In the case of Et4N[H3(BAr(F)3)2], strong acids such as HCl induce H2 release to give the chloride Et4N[(CO)(CNBAr(F)3)2Fe(Cl)(pdt)Ni(dppe)].

SUBMITTER: Manor BC

PROVIDER: S-EPMC3843950 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Hydrogen activation by biomimetic [NiFe]-hydrogenase model containing protected cyanide cofactors.

Manor Brian C BC Rauchfuss Thomas B TB

Journal of the American Chemical Society 20130730 32

Described are experiments demonstrating incorporation of cyanide cofactors and hydride substrate into [NiFe]-hydrogenase (H2ase) active site models. Complexes of the type (CO)2(CN)2Fe(pdt)Ni(dxpe) (dxpe = dppe, 1; dxpe = dcpe, 2) bind the Lewis acid B(C6F5)3 (BAr(F)3) to give the adducts (CO)2(CNBAr(F)3)2Fe(pdt)Ni(dxpe), (1(BAr(F)3)2, 2(BAr(F)3)2). Upon decarbonylation using amine oxides, these adducts react with H2 to give hydrido derivatives [(CO)(CNBAr(F)3)2Fe(H)(pdt)Ni(dxpe)](-) (dxpe = dppe ...[more]

PMID: 23899049

Similar Datasets

Project description:BACKGROUND:Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii "Deep ecotype" that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. RESULTS:We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. CONCLUSIONS:Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.

Project description:Fatty acids produced by H2-metabolizing bacteria are sometimes observed to be more D-depleted than those of photoautotrophic organisms, a trait that has been suggested as diagnostic for chemoautotrophic bacteria. The biochemical reasons for such a depletion are not known, but are often assumed to involve the strong D-depletion of H2. Here, we cultivated the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha H16) under aerobic, H2-consuming, chemoautotrophic conditions and measured the isotopic compositions of its fatty acids. In parallel with the wild type, two mutants of this strain, each lacking one of two key hydrogenase enzymes, were also grown and measured. In all three strains, fractionations between fatty acids and water ranged from -173‰ to -235‰, and averaged -217‰, -196‰, and -226‰, respectively, for the wild type, SH- mutant, and MBH- mutant. There was a modest increase in ?D as a result of loss of the soluble hydrogenase enzyme. Fractionation curves for all three strains were constructed by growing parallel cultures in waters with ?Dwater values of approximately -25‰, 520‰, and 1100‰. These curves indicate that at least 90% of the hydrogen in fatty acids is derived from water, not H2. Published details of the biochemistry of the soluble and membrane-bound hydrogenases confirm that these enzymes transfer electrons rather than intact hydride (H-) ions, providing no direct mechanism to connect the isotopic composition of H2 to that of lipids. Multiple lines of evidence thus agree that in this organism, and presumably others like it, environmental H2 plays little or no direct role in controlling lipid ?D values. The observed fractionations must instead result from isotope effects in the reduction of NAD(P)H by reductases with flavin prosthetic groups, which transfer two electrons and acquire H+ (or D+) from solution. Parallels to NADPH reduction in photosynthesis may explain why D/H fractionations in C. necator are nearly identical to those in many photoautotrophic algae and bacteria. We conclude that strong D-depletion is not a diagnostic feature of chemoautotrophy.

Dataset Information

Hydrogen activation by biomimetic [NiFe]-hydrogenase model containing protected cyanide cofactors.

Publications

Hydrogen activation by biomimetic [NiFe]-hydrogenase model containing protected cyanide cofactors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets