Characterization of the oxygen tolerance of a hydrogenase linked to a carbon monoxide oxidation pathway in Rubrivivax gelatinosus.

Hydrogen is a clean fuel that addresses both adverse environmental impacts and periodically recurring energy crises. Industrial attention has been drawn to the microbial production of H2 using hydrogenase enzymes because of the wide variety of acceptable feedstock which could substantially lower the cost of H2 production. Hydrogenases catalyze the reversible reduction of protons into H2, although most are committed to catalyze either in the H2 oxidation direction or in the H2 evolution direction based on the energy demands of the cells. This explains the existence of multiple hydrogenases within a living organism, each expressed under certain physiological conditions so that the organism can best meet its energy need (2, 3, 20). Potential applications of photosynthetic and fermentative microorganisms in the generation of H2 have been reviewed extensively (9, 13, 29). Among these microorganisms, certain photosynthetic bacteria are unique in that they contain a CO oxidation pathway converting CO and H2O into H2 and CO2. This reaction has been reported for Rubrivivax gelatinosus (19, 24, 25), Rhodospirillum rubrum (6, 20, 25), and Rhodopseudomonas palustris (16). Carbon monoxide is demonstrated to induce the de novo synthesis of a hydrogenase enzyme linking to the CO oxidation pathway in Rhodospirillum rubrum (7). An ideal process to produce H2 more economically would be water derived. Lacking photosystem II, photosynthetic bacteria cannot use water as the electron donor. Cyanobacteria, on the other hand, have both photosystems I and II and can therefore oxidize water to generate photoreductants. One potential solution is to express bacterial hydrogenase along with its physiological electron mediator(s) in a cyanobacterial host. However, most H2-evolving hydrogenases are extremely sensitive to O2, which is an inherent byproduct of cyanobacterial photosynthesis. This sensitivity to O2 was noted in reports of hydrogenases from Clostridium pasteurianum, Desulfovibrio vulgaris, and Chlamydomonas reinhardtii, where 50% of the initial evolution activities were lost from the reduced enzymes within several minutes of exposure to O2 (1, 2, 11). Therefore, to establish a successful cyanobacterium-bacterium hybrid system using H2O as the electron donor, one critical requirement is to use a hydrogenase that is not only tolerant to O2 but also catalytically active in O2. The photosynthetic bacterium Rubrivivax gelatinosus CBS contains an active CO-to-H2 pathway (19), and this report documents the O2 tolerance displayed by its CO-linked hydrogenase. Besides using reduced methyl viologen (MV) to measure hydrogenase activity, which precludes the addition of O2 during the assay, it is ultimately important for us to determine whether this hydrogenase can also function in the simultaneous presence of O2. Most hydrogenases catalyze a hydrogen- deuterium (H-D) exchange reaction in a hydrogen-deuterium oxide (D2O) system, yielding HD (12, 15, 21, 30), and the isotopic exchange rate correlates positively with the H2 production rate mediated by reduced MV (21). Because this reaction does not require a reducing agent such as sodium dithionite, O2 can be included throughout the assay without being scavenged by the reducing agent. Since this assay does not involve any electron mediator, it becomes a direct assay of the enzyme itself. Consequently, any effect O2 might have on the H-D exchange activity reflects the intrinsic nature of the enzyme. In this report, we describe findings of how O2 affected the rate of the exchange reaction and its reversibility upon the removal of O2. We also determined the sensitivity of this hydrogenase to CO once the bulk of the CO dehydrogenase (CODH) activity was removed. Finally, we constructed a spinach-ferredoxin-hydrogenase system and discuss its implication for the generation of H2 from a cyanobacterium-bacterium hybrid system.