(620f) Proteomic Analysis of a Hydrogen Producing Thermophile Carboxydothermus Hydrogenoformans

The development of an economic means of hydrogen production is necessary before H2 can be widely implemented as a replacement for fossil fuels. Currently, the majority of H2 produced in the US is generated through steam reforming of natural gas or coal gasification, both energy intensive processes. A limited number of microorganisms have been identified which are capable of producing H2, utilizing CO as their sole carbon and energy source. To fully utilize and optimize microbial H2 production, it is necessary to understand how the CO metabolic process functions. One thermophilic carboxydotroph, Carboxydothermus hydrogenoformans, was chosen as a model organism for studies of carbon monoxide metabolism employing proteomic methodologies. Protein expression was compared under different growth conditions: (1) 0.05 g/L versus 0.5 g/L yeast extract, and (2) AQDS CO free versus pure CO headspace. Samples were harvested under anaerobic conditions. For protein expression analysis, the cells were disrupted via a French pressure cell and membrane and soluble fractions were separated by ultracentrifugation. Fractions from paired samples were digested with trypsin and isobarically labeled prior to LC separation. Peptides fractions were spotted onto MALDI plates and subjected to MS/MS analyses. An initial sample of cells grown at the 0.5 g/L yeast extract concentration was analyzed to confirm that different CODH protein subunits were detectable. Analysis of the MS/MS data from the soluble fraction identified peptides corresponding to over 15 different proteins from C. hydrogenoformans (p<0.05). Two of the proteins identified were the cooS II (CHY_0085) and cooS IV (CHY_1824) CODH subunits. Four peptides from CHY_0085 and three from CHY_1824 were identified with significant probability. Studies of the membrane bound proteins and labeled samples are currently underway.