CYTOCHROME C(553), FROM DESULFOVIBRIO-VULGARIS (HILDENBOROUGH) - ELECTROCHEMICAL PROPERTIES AND ELECTRON-TRANSFER WITH HYDROGENASE

An electrochemical study of the periplasmic cytochrome c(553) of Desulfovibrio vulgaris (Hilden-borough) is presented. The dependence of the midpoint potential on temperature and pH was studied with cyclic voltammetry. The voltammograms obtained were reversible and revealed that this cytochrome showed fast electron transfer on a bare glassy carbon electrode. The midpoint potential at pH 7.0 and 25 degrees C was found to be 62 mV versus the normal hydrogen electrode. It was observed that the temperature dependence was discontinuous with a transition temperature at 32 degrees C. The standard reaction entropy at the growth temperature of the organism (37 degrees C) was calculated to be Delta S degrees' = -234 J mol(-1) K-1. The pH dependence of the midpoint potential could be described with one pK of the oxidized form with a value of 10.6. The second-order rate constant for electron transfer between cytochrome c(553) and the Fe-hydrogenase from D. vulgaris (H) was also determined with cyclic voltammetry. The equivalent rate constant for cytochrome c(3) and hydrogenase was measured for comparison. The second-order rate constants are 2X10(7)M(-1) s(-1) for cytochrome c(553) and 2X10(8)M(-1) s(-1) for cytochrome c(3). The kinetic parameters of the hydrogenase for both cytochromes were determined using the spectrophotometric hydrogen consumption assay. With cytochrome c(553) this resulted in a K-m of 46 mu M and a maximum turnover number of 7.1x10(2) s(-1) in the H-2 consumption assay. The values with cytochrome c(3) were 17 mu M and 6.4X10(2) s(-1), respectively. The importance of the different kinetic parameters for contrasting models proposed to describe the function of the Fe-hydrogenase are discussed.