A VARIABLE-TEMPERATURE DIRECT ELECTROCHEMICAL STUDY OF METALLOPROTEINS FROM HYPERTHERMOPHILIC MICROORGANISMS INVOLVED IN HYDROGEN-PRODUCTION FROM PYRUVATE

The hyperthermophilic bacterium Thermotoga maritima and the hypertherm ophilic archaeon Pyrococcus furiosus grow optimally at 80 and 100 degr ees C, respectively, by the fermentation of carbohydrates to organic a cids, CO2, and H-2. Pyruvate is a major source of reductant for H-2 pr oduction during fermentation, and pyruvate ferredoxin oxidoreductase ( POR), a 4Fe-type ferredoxin, and hydrogenase have been previously puri fied from both species. P. furiosus utilizes a copper-iron-containing POR and a nickel-iron-containing hydrogenase, whereas the POR of T. ma ritima lacks copper and its hydrogenase lacks nickel. For all four enz ymes and for the two ferredoxins, we have determined their reduction p otentials (E degrees') and, where possible, thermodynamic parameters a ssociated 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 al l six proteins were comparable and spanned less than 50 mV, but their temperature dependence varied dramatically, even between analogous pro teins, such that in the physiological-relevant temperature range the E degrees' values became widely separated. In most cases, transition po ints were observed in E degrees'/temperature profiles, and these gener ally corresponded with significant increases in catalytic activity, bu t occurred at lower temperatures in T. maritima than in P. furiosus. T he two ferredoxins (and also P. furiosus rubredoxin) had much more neg ative entropy terms than were calculated for POR and hydrogenase, and these values were also more negative than those previously reported fo r 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. The results show that the electron transport pathways between POR and hydrogenase are very different in these two hyperthermophilic organis ms. It is concluded that reduction potentials measured at ambient temp erature appear to be of little value in rationalizing electron transfe r processes in hyperthermophilic proteins and in fact may be quite mis leading.