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

Abstract

Spectroscopic and potentiometric measurements have been carried out, at room temperature, during anaerobic titrations of Hansenula anomala L-lactate cytochrome c oxidoreductase (or flavocytochrome b,) both in the presence and in the absence of pyruvate (the physiological reaction product). Under the same conditions, the flavin spectral contribution was estimated and the flavosemiquinone proportion was directly determined by electron paramagnetic resonance measurements.In the present study, we show the visible light absorption and paramagnetic characteristics of the flavin radical at 18°C and also the dramatic effect of pyruvate on the redox potential of each monoelectronic couple of the flavin. Thermodynamic stabilization of the semiquinone form, in the presence of pyruvate, is interpreted as a mode of regulation of flavocytochrome b2 activity. Taking into account that analogous controls have been observed with two other flavoenzymes belonging to this class of dehydrogenases/one-electron transferases, we suggest that redox potential modulation could be a type of regulation effective for the whole class of enzymes in which a semiquinone is an obligate intermediate.Flavoenzymes differ from other redox proteins of oxidative chains in that their prosthetic group does not shuttle between two redox states, as is the case of NAD(P)H, ironsulfur centers and iron porphyrin, but between three redox states: oxidized, semiquinone and fully reduced, i. e. hydroquinone. These three states, as underlined by Hemmerich and Massey 111, are not all involved in the normal mechanism of each flavoprotein; they are thus in the class of dehydrogenases/one-electron transferases in which the coupling between 2e-donors and le-acceptors requires the active role of the semiquinone intermediate in the r e d o n sequence during turnover.We suggest that a general procedure of feedback control in dehydrogenases/le-transferases might involve modulation of the redox parameters defining equilibrium proportions of the three flavin states by preferential binding of the reaction product to one of them. Indeed, as shown in the present paper, we observed in a yeast mitochondria1 flavocytochrome enzyme, L-lactate : cytochrome c oxidoreductase, that pyruvate, the reaction product, binds preferentially to the semiquinone form with concomitant large shifts of the midpoint potentials corresponding to the two one-electron flavin couples. 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 ...

Studies on the Redox Centers of the Terminal Oxidase fromDesulfovibrio gigas and Evidence for Its Interaction with Rubredoxin

“…Since the spectral features remain unaltered in a broad temperature range (20 -200 K for EPR and 77-298 K for visible spectroscopy), it may be concluded that they are low-spin in this entire temperature range. Two other low intensity resonances are also observed in some samples: a minor component at g ϳ 6, characteristic of high-spin hemes, and a radical type signal at g ϳ 2.0, with a line width of 1.6 mT, identical to those observed in flavin red semiquinones (49).…”

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

“…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. The assumption that this complex would behave as a dead-end complex was proposed [2]; we suggested that this behaviour could be a general mode of control of the activity of dehydrogenases/one-electron-transferases towards external acceptors. The object of the present study was to characterize further the behaviour of pyruvate with respect to flavocytochrome h2.…”

“…In particular, it reaches nearly 94 F 4% of the total flavin in the presence of 10mM pyruvate [2]. This effect a priori corresponds to a preferential binding of pyruvate to the F, species relative to F, or Fh.…”

“…Clark [17]). (b) A dual-wavelength set 460/544 nm was used to monitor the (Fh + F,) + 2 F, transition on the basis of studies detailed in [2]. Since the increments of the differential absorbance upon pyruvate addition are then a linear function of [F,], the corresponding calibration curve (not shown) is well defined by three points at 0, 1 and 10 mM pyruvate [18] for which [F,] has been precisely determined by EPR spectrometry [2].…”

“…cerevisiae enzyme [14]. [2]. After each addition of pyruvate with a microsyringe, the level of heme reduction was checked and, when necessary, reset to 62% by photoreduction, then absorption variation was recorded at seven wavelengths (600, 573, 556, 544, 490 and 460nm) using a wavelength program.…”