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

Abstract

Temperature-jump experiments on flavocytochrome b, 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.At pH 7.0 the mean reciprocal relaxation time is l/z* = 190 s -', 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 (7-' = 2500 s-I), predominant at high percentages of reduced heme, has been assigned to the reduction of heme by flavin hydroquinone, while the slower process (7-' = 350 s-'), 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. A great deal of information, essentially based on rapidmixing experiments, is available on the mechanism and the kinetic parameters of the reactions of the prosthetic groups with external donors and acceptors, for both the enzymes purified from Saccharomyces cerevisiue [14,17] Since the prosthetic groups have redox potentials close to each other and rapidly reach equilibrium at partial reduction in the absence of oxygen [15, 181, relaxation techniques appear suitable to attack the problem. In this paper, we report a series of temperature-jump experiments aimed at studying the intramolecular electron transfer carried out mainly at pH 7.0, where most of the steps of the enzyme catalytic cycle are well characterized. Preliminary experiments at pH 6.0 are also presented. In view of the known reactivity of flavocytochrome b, towards oxygen [19,20], anaerobic conditions were used. Since the product of the physiological reaction, i.e. pyruvate, is known to yield a mixed-type inhibition [21] and to form a complex with the oxidized enzyme [22], the relaxation behaviour in the presence of pyruvate at pH 7.0 has also been investigated. MATERIALS AND METHODSFlavocytochrome b, was purified from Hansenulu unomala yeast essentially as described in [I] with the modifications detailed in [23]. The resulting enzyme, homogeneous in sodium dodecyl sulfate electrophoresis, has a ratio A,,,/A,,, (reduced) = 0.490 and a standard molar activity [I] at 30°C equal to 1000s-' (mole electron equivalent per mole of enzyme protomer). This value becomes 500 s-' when expressed as mole of substrate per mole of enzyme protomer. Preparations were stocked at 4 "C as suspensions in 50 % saturated ammonium sulfate containing 50 m M Dr.-lactate, 100 mM phosphate buffer, pH 7. Un...

Inhibition of L‐lactate: cytochrome‐c reductase (flavocytochrome b2) by product binding to the semiquinone transient

“…In 1984 [l, 21, we observed similar changes in redox equilibria in the case of the yeast L-lactate cytochrome c oxidoreductase (flavocytochrome b 2 ) in the presence of pyruvate (as reported in Table 4); the rate constants estimated in temperature-jump studies [3] were also modified (Table 2). Indeed, for the oneelectron transfer from the flavosemiquinone to oxidized heme, the gap between the relevant midpoint potentials ( A Em, acceptor minus donor systems) becomes negative in the transient pyruvate-liganded enzyme so that the transfer is thermodynamically unfavorable.…”

“…In [3], the k , and k -values were calculated from the relaxation times and the values of equilibrium constants available in 1984 [7]. The…”

Regulation of dehydrogenases/one‐electron transferases by modification of flavin redox potentials

“…This leads to a strong increase in the level of flavosemiquinone and inhibition of activity. This effect of pyruvate on the equilibrium proportion of flavin semiquinone and also on individual rate constants in the intramolecular electron exchange was first detected in the course of a temperature-jump investigation in collaboration with Brunori and Silvestrini [2].Two other similar observations have been previously reported concerning the behaviour of succinate dehydrogenase in the presence of oxaloacetate [3, 41 and of NADH:cytochrome b, reductase in the presence of NAD' [S, 61. In these flavoenzymes, the 'product' binding that takes place preferentially to the oxidized and to the fully reduced forms, respectively, modifies the redox potentials of the bielectronic flavin couple and stabilizes enzyme complexes that depress turnover activity.…”

“…The values listed in Table 1 for K 1 , in the absence and in the presence of pyruvate (that is Kl,,) lead to the estimate KP,,, = 0.8 mM taking into consideration the value of K,,,, = 8 mM (Tegoni, unpublished). A rough estimation of this affinity constant has been made on the basis of temperature-jump experiments [2]: in fact z-l values for the intramolecular electron exchange between F,, and H,, drastically change in the presence of pyruvate; the maximal effect, in this case, has already been detected for a concentration of 1 mM pyruvate. The estimation of the same constant, in an experiment at variable concentration of pyruvate (not shown), leads to a value of about 0.4 mM.…”

“…This leads to a strong increase in the level of flavosemiquinone and inhibition of activity. This effect of pyruvate on the equilibrium proportion of flavin semiquinone and also on individual rate constants in the intramolecular electron exchange was first detected in the course of a temperature-jump investigation in collaboration with Brunori and Silvestrini [2].…”