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Probing intermediates in the activation cycle of [NiFe] hydrogenase by infrared spectroscopy: the Ni-SIr state and its light sensitivity.

ABSTRACT: The [NiFe] hydrogenase from the sulphate-reducing bacterium Desulfovibrio vulgaris Miyazaki F is reversibly inhibited in the presence of molecular oxygen. A key intermediate in the reactivation process, Ni-SI(r), provides the link between fully oxidized (Ni-A, Ni-B) and active (Ni-SI(a), Ni-C and Ni-R) forms of hydrogenase. In this work Ni-SI(r) was found to be light-sensitive (T

SUBMITTER: Pandelia ME

PROVIDER: S-EPMC2847147 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Probing intermediates in the activation cycle of [NiFe] hydrogenase by infrared spectroscopy: the Ni-SIr state and its light sensitivity.

Pandelia Maria-Eirini ME Ogata Hideaki H Currell Leslie J LJ Flores Marco M Lubitz Wolfgang W

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 20090721 8

The [NiFe] hydrogenase from the sulphate-reducing bacterium Desulfovibrio vulgaris Miyazaki F is reversibly inhibited in the presence of molecular oxygen. A key intermediate in the reactivation process, Ni-SI(r), provides the link between fully oxidized (Ni-A, Ni-B) and active (Ni-SI(a), Ni-C and Ni-R) forms of hydrogenase. In this work Ni-SI(r) was found to be light-sensitive (T <or= 110 K), similar to the active Ni-C and the CO-inhibited states. Transition to the final photoproduct state (Ni-S ...[more]

PMID: 19626348

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Project description:BackgroundPhotosynthetic 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.ResultsWe 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.ConclusionsOur 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.

Dataset Information

Probing intermediates in the activation cycle of [NiFe] hydrogenase by infrared spectroscopy: the Ni-SIr state and its light sensitivity.

Publications

Probing intermediates in the activation cycle of [NiFe] hydrogenase by infrared spectroscopy: the Ni-SIr state and its light sensitivity.

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