PURIFICATION AND PROPERTIES OF A NOVEL CYTOCHROME - FLAVOCYTOCHROME-CFROM SHEWANELLA-PUTREFACIENS

The major soluble cytochrome isolated from microaerobically grown cell s of Shewanella putrefaciens has been shown to be a novel type of flav ocytochrome with fumarate reductase activity. This flavocytochrome, lo cated in the periplasmic fraction of cell extracts, has been purified to homogeneity and shown to contain 4 mol of haem c and 1 mol of non-c ovalently bound FAD per mol of protein. An M(r) value of 63 800 is est imated from sequence analysis assuming 4 mol of haem/mol of protein. I n the presence of the artificial electron donor, reduced methyl violog en, the flavocytochrome catalysed the reduction of fumarate but not th at of nitrite, dimethylsulphoxide, trimethylamine-N-oxide or sulphite. The pH optimum was 7.4 with calculated pK(a) values of 6.8 and 8.0 fo r contributing catalytic groups. The K-m and k(cat) values for fumarat e reduction were 21 mu M and 250 s(-1) respectively, whereas the corre sponding values for succinate oxidation with 2,6-dichlorophenol-indoph enol as electron carriers were 200 mu M and 0.07 s(-1) respectively. M esaconic acid was a competitive inhibitor of fumarate reduction with a K-i of 2 mu M. Zymogram staining of polyacrylamide gels with purified protein showed a band of fumarate reductase activity. Polyclonal anti bodies, raised to the purified flavocytochrome, were shown to titrate out fumarate reductase activity. We conclude that the physiological ro le of this enzyme is as a fumarate reductase. Optical absorption spect ra of the flavocytochrome indicated that all the haems were of the c-t ype and gave alpha, beta and gamma peaks at 552.3, 523 and 418 nm in t he reduced spectrum with epsilon values of 30.2, 15.9 and 188.2 mM(-1) cm(-1) respectively. Oxidized spectra showed no 695 nm band that woul d be indicative of His-Met coordination. Two redox potentials were res olved at -220 mV and -320 mV. The cytochrome was reduced by formate in the presence of particulate cell fractions. The relationship of this cytochrome to other low-potential flavocytochromes c is discussed.