A temperature-jump study of the electron transfer reactions in Hansenula anomala flavocytochrome b2

Temperature-jump experiments on flavocytochrome b2 were carried out at different levels of heme reduction at pH 7.0 and 6.0, and as a function of pyruvate concentration. The relaxation, corresponding to an increase in the concentration of reduced heme, is in no case a simple process. AtpH 7.0 the mean reciprocal relaxation time is 1/tau* = 190 s-1, independent of enzyme concentration, wavelength of observation and percentage of heme reduction. Flavin semiquinone has been identified as the major electron donor to the heme in this process. At the same pH the presence of pyruvate in the millimolar concentration range increases the relaxation rate and affects its amplitude. The latter effect could be accounted for by a change in redox equilibria between heme and flavin upon pyruvate binding. At pH 6.0 the relaxation pattern depends more clearly on the level of heme reduction. A rapid process (tau-1 = 2500 s-1), predominant at high percentages of reduced heme, has been assigned to the reduction of heme by flavin hydroquinone, while the slower process (tau-1 = 350 s-1), essentially the only one present at or below 50% of heme reduction, has been ascribed to the reduction of heme by flavin semiquinone. These results are discussed in relation to the catalytic mechanism of the enzyme.