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Characterization of the Oxygen Tolerance of a Hydrogenase Linked to a Carbon Monoxide Oxidation Pathway in Rubrivivax gelatinosus

The National Renewable Energy Laboratory, Golden, Colorado 80401-3393

Received 14 December 2001/ Accepted 7 March 2002

A hydrogenase linked to the carbon monoxide oxidation pathway in Rubrivivax gelatinosus displays tolerance to O₂. When either whole-cell or membrane-free partially purified hydrogenase was stirred in full air (21% O₂, 79% N₂), its H₂ evolution activity exhibited a half-life of 20 or 6 h, respectively, as determined by an anaerobic assay using reduced methyl viologen. When the partially purified hydrogenase was stirred in an atmosphere containing either 3.3 or 13% O₂ for 15 min and evaluated by a hydrogen-deuterium (H-D) exchange assay, nearly 80 or 60% of its isotopic exchange rate was retained, respectively. When this enzyme suspension was subsequently returned to an anaerobic atmosphere, more than 90% of the H-D exchange activity was recovered, reflecting the reversibility of this hydrogenase toward O₂ inactivation. Like most hydrogenases, the CO-linked hydrogenase was extremely sensitive to CO, with 50% inhibition occurring at 3.9 µM dissolved CO. Hydrogen production from the CO-linked hydrogenase was detected when ferredoxins of a prokaryotic source were the immediate electron mediator, provided they were photoreduced by spinach thylakoid membranes containing active water-splitting activity. Based on its appreciable tolerance to O₂, potential applications of this hydrogenase are discussed.

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• Maness, P.-C., Huang, J., Smolinski, S., Tek, V., Vanzin, G. (2005). Energy Generation from the CO Oxidation-Hydrogen Production Pathway in Rubrivivax gelatinosus. Appl. Environ. Microbiol. 71: 2870-2874 [Abstract] [Full Text]