Dr. Michael Seibert
National Renewable Energy Laboratory
Unknown Institution
Golden, CO

Wednesday, May 28, 2003
12:10 am (CT)
3169 Beckman Institute

Abstract

Sulfur deprivation of Chlamydomonas reinhardtii cultures gradually inactivates photosynthetic O2-evolution capacity in the algae to levels below that of O2 consumption by cellular respiration. At that point anaerobiosis is established, and the in situ photochemical activity of photosystem II (PSII, the light reaction associated with O2 evolution) abruptly drops to ca. zero. Shortly thereafter, PSII activity reappears, and then H2 photoproduction commences as shown at the right. The abrupt loss of photochemical activity results from the over-reduction of the plastoquinone (PQ) pool (electron transport carriers located between PSII and PSI, the light reaction associated with H2 production), caused by the lack of both O2 and other pathways available to re-oxidize the PQ-pool. Once hydrogenase (the O2-sensitive enzyme that releases H2) activity is induced, over-reduction of the PQ-pool is relieved, partial recovery of PSII photochemical activity is observed, and H2 can be collected. Since most of the electrons used directly by the algae for H2 photoproduction originate from water oxidation by residual PSII activity, the redox state of the PQ-pool serves a regulatory function in the system. Given that PSII activity, even at reduced levels, results in the co-production of some O2, sulfur-deprived cells must have a mechanism to dispose of the gas, since hydrogenase activity requires the continued presence of an anaerobic environment. Mitochondrial respiration rather chlororespiration seems to be involved in this process, and storage products such as starch and protein provide the substrate. The original batch process (figure) was first reported three years ago, and recent improvements that allow continuous H2-gas production will be examined. The sulfur-deprivation process works because the nutrient stress reduces the activity of PSII by about a factor of ten. However, an applied algal H2-production process should operate at the full efficiency of photosynthesis, and this will require addressing the hydrogenase O2-sensitivity problem. Recent results have now identified two expressed algal hydrogenases, HydA1 and HydA2, which may be involved in the H2-photoproduction process. Simplistic structural modeling of their amino acid sequences suggests that O2 gains access to the active site (an unusual Fe-S cluster) through a channel that functions in H2 diffusion from the protein. Molecular engineering of HydA1 to restrict the access of O2 through the H2 channel to the catalytic site has now demonstrated that is possible to improve the O2 tolerance of the enzyme. Future prospects for further improvement will be examined.