Comparison of the processes involved in reduction by the substrate for two homologous flavocytochromes b2 from different species of yeast.

A detailed study of the electron exchanges involved between FMN and haem b2 groups within flavocytochrome b2 of yeast Hansenula anomala (H-enzyme) was performed. The results were compared with those for the homologous enzyme of yeast Saccharomyces cerevisiae (Sx-enzyme) re-investigated at 5 degrees C. The mid-point reduction potentials of FMN and haem were determined by two complementary methods: potentiometric titration with substrate, L-lactate, in the presence of dye mediators with quantification of the reduced species performed by spectrophotometry at suitable wavelengths; anaerobic titration of the enzyme by its substrate by monitoring the e.p.r. signals of the semiquinone and Fe3+ species. Values of Em,7 = -19, -23 and -45 V were determined respectively from the data for the three redox systems Ho/Hr, Fo/Fsq and Fsq/Fr in the H-enzyme instead of +6, -44 and -57 mV respectively in the Sx-enzyme [Capeillère-Blandin, Bray, Iwatsubo & Labeyrie (1975) Eur. J. Biochem. 54, 549-566]. Parallel e.p.r rapid-freezing and absorbance stopped-flow studies allowed determination of the time courses of the various redox species during their reduction by L-lactate. The flavin and the haem reduction time courses were biphasic. In the initial fast phase the reduction of flavin monitored by absorbance measurements is accomplished with a rate constant kF = 360 s-1. The reduction of the haem lags the reduction of flavin with a rate constant kH = 170 s-1. The appearance of flavin free radical is slower than the reduction in flavin absorbance and occurs with a rate constant close to that of the reduction of the haem. At saturating L-lactate concentration the initial rapid phase (up to 15 ms) involved in the overall turnover can be adequately simulated with a two-step reaction scheme. The main difference between the enzymes lies especially at the level of the first step of electron exchange between bound lactate and flavin, which for the H-enzyme is no longer the rate-limiting step in the haem reduction and becomes 8-fold faster than in the Sx-enzyme. Consequently in the H-enzyme for the following step, the intramolecular transfer from flavin hydroquinone to oxidized haem, a reliable evaluation of the rate constants becomes possible. Preliminary values are k+2 = 380 s-1 and k-2 = 120 s-1 at 5 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)