Semiquinones flavocytochrome

Electron flow through flavocytochrome bz has been extensively studied in both the S. cerevisiae (Tegoni et al., 1998 Daff et al., 1996a Chapman et al., 1994 Pompon, 1980) and H. anomala (CapeillEre-Blandin et al., 1975) enzymes. The catalytic cycle is shown in Figure 3. Firstly, the flavin is reduced by L-lactate a carbanion mechanism has been proposed for this redox step (Lederer, 1991). Complete (two-electron) reduction of the flavin is followed by intra-molecular electron transfer from fully-reduced flavin to heme, generating flavin semiquinone and reduced heme (Daff et al.. 

FIGURE 3. The Catalytic Cycle for Flavocytochrome 62 F, flavin H, heme Cyt c, cytochrome c. Electrons are shown as hlack dots and are used to indicate the two-electron reduced flavin (hydroquinone), F with two dots one-electron reduced flavin (semiquinone), F with one dot reduced heme, H with one dot reduced cytochrome c, Cyt c with one dot. The rate constants shown are for S. cerevisiae flavocytochrome hi at 25 C, pH 7.5, I = O.IOM. The whole catalytic cycle turns over at approximately 100 s ". The details of the cycle are described in the main text. 

Whiehever mechanism operates, it is clear that the rate of reduetion of the flavin group is totally limited by the cleavage of the aC-H bond sinee the deuterium kinetic isotope effect for this step is around 8 (Miles et al., 1992 Pompon et al., 1980). However, in flavocytochrome 2 the rate of flavin reduetion is some 6-fold faster than the overall steady-state turnover rate (Daff et al., 1996a). As a consequence the flavin reduction step eontributes little to the rate limitation of the overall catalytic cycle (Figure 3). In faet it is eleetron transfer from flavin-semiquinone to b2 -heme that is the major rate-determining step and this is discussed in the following seetion. 

EPR signals for both the flavosemiquinone radical and the low-spin ferric heme have been reported (65, 78-82). The flavosemiquinone signal, which is easily observed at 123 K, shows a typical g value of 2.0039 0.002 (65). The bandwidth, which is around 15 G, is very like that of an anionic, or red, semiquinone (65). The EPR signal of the low-spin ferric heme can be observed at low temperatures ( 28 K) and shows g values of 2.99, 2.22, and 1,47 (65), which are similar to those found for cytochrome 65 (81). EPR rapid freezing studies have allowed the amounts of semiquinone and ferric heme to be monitored during reduction of the enzyme by L-lactate (66). This has proved to be extremely useful in the development of kinetic schemes to describe the flow of electrons in the enzyme. The distance between the prosthetic groups in H. anomala flavocytochrome 62 has been estimated from EPR experiments and spin-lattice relaxation measurements (82). Pyruvate was used to stablize the flavosemiquinone and the effect on the signal of this species from oxidized and reduced heme was measured. The results indicated a minimum intercenter distance of 18-20 A (82). 

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