A variable-temperature direct electrochemical study of metalloproteins from hyperthermophilic microorganisms involved...

The hyperthermophilic bacterium Thermotoga maritima and the hyperthermophilic archaeon Pyrococcus furiosus grow optimally at 80 and 100 degrees C, respectively, by the fermentation of carbohydrates to organic acids, CO2, and H2. Pyruvate is a major source of reductant for H2 production during fermentation, and pyruvate ferredoxin oxidoreductase (POR), a 4Fe-type ferredoxin, and hydrogenase have been previously purified from both species. P. furiosus utilizes a copper-iron-containing POR and a nickel-iron-containing hydrogenase, whereas the POR of T. maritima lacks copper and its hydrogenase lacks nickel. For all four enzymes and for the two ferredoxins, we have determined their reduction potentials (E degrees') and, where possible, thermodynamic parameters associated with electron transfer (delta S degrees and delta H degrees), using differential pulse voltammetry at temperatures ranging from 25 to 95 degrees C. At ambient temperature, the E degrees' values for all six proteins were comparable and spanned less than 50 mV, but their temperature dependence varied dramatically, even between analogous proteins, such that in the physiological-relevant temperature range the E degrees' values became widely separated. In most cases, transition points were observed in E degrees'/temperature profiles, and these generally corresponded with significant increases in catalytic activity, but occurred at lower temperatures in T. maritima than in P. furiosus. The two ferredoxins (and also P. furiosus rubredoxin) had much more negative entropy terms than were calculated for POR and hydrogenase, and these values were also more negative than those previously reported for mesophilic redox proteins. The reduction potentials measured at high temperatures and likely efficiencies of electron transfer between the various proteins were consistent with in vitro activity measurements.(ABSTRACT TRUNCATED AT 250 WORDS)