ELECTRON-TRANSFER FROM PHANEROCHAETE-CHRYSOSPORIUM CELLOBIOSE OXIDASE TO EQUINE CYTOCHROME-C AND PSEUDOMONAS-AERUGINOSA CYTOCHROME C-551

The electron-transfer reactions of cellobiose oxidase (CBO) have been investigated by conventional and by rapid-scan stopped-flow spectroscopy at pH 6.0. Analysis of the absorbance/time/ wavelength matrix by Singular Value Decomposition (SVD) confirms earlier studies showing that cellobiose rapidly reduces the flavin group (7.7 s(-1); cellobiose, 100 mu M) which in turn slowly (0.2 s(-1)) reduces the cytochrome b moiety. In the presence of CBO, cellobiose reduces cytochromes c in a reaction that does not depend on oxygen or superoxide. The rate limit for this process is independent of the source of the cytochromes c and is identical with the rate of cytochrome b reduction. Rapid-mixing experiments show that cytochrome b may donate electrons very rapidly to either mammalian cytochrome c or bacterial cytochrome c-551. The reactions were second-order (k(c) = 1.75 x 10(7) M(-1).s(-1); k(c-551) = 4.3 x 10(6) M(-1).s(-1); pH 6.0, 21 degrees C and 10.064) and strongly ionic-strength (I)-dependent: k(c) decreasing with I and k(c-551) increasing with I. These results suggest the electron-transfer site near cytochrome b bears a significant negative charge. Equilibrium gel chromatography confirms that CBO oxidase and positively charged mammalian cytochrome c make stable complexes. These results are discussed in terms of a model suggesting an electron-transfer role for cytochrome b in vivo, possibly connected with radical-mediated cellulose breakdown.