Étude par dichroïsme circulaire des interactions hème‐flavine‐protéine dans le cytochrome <i>b</i><sub>2</sub> (<scp>l</scp>‐lactate oxydoréductase) de la levure

European Journal of Biochemistry

1978

Baker's yeast L-lactate dehydrogenase (flavocytochrome b2) is a typical flavodehydrogenase, in that it accepts two electrons from the substrate but has a monoelectronic acceptor. Yet it forms a red semiquinone [Capeillere Blandin et al. Eur. J. Bioclwm. 54, 549-566 (1975)j and it is shown in this paper that it forms a reversible covalent complex with sulfite (Kd = 1.4 pM). This complex can be observed by difference spectroscopy and provides a convenient tool for visualizing the flavin chromophore, usually hidden behind the intense heme absorbance. A number of anions (D-lactate, oxalate and pyruvate) are inhibitors of the enzymatic reaction and induce spectral perturbations of the flavin spectrum. It is concluded that probably two positive charges exist at the active site: one which stabilizes the red semiquinone and one which attracts organic anions and sulfite. It is also concluded that the correlation between reactivity with sulfite and reactivity with oxygen among flavoproteins may not be as general as previously proposed [Massey et al. J. Biol. Chem. 244,[3999][4000][4001][4002][4003][4004][4005][4006] (1 969)].Free reduced flavin is rapidly reoxidized by oxygen, whereas enzyme-bound flavin either keeps or loses this property, according to the function of the protein. This phenomenon provides the basis for the now classical distinction, among flavoproteins, between oxidases and dehydrogenases. It is clear that the protein moiety plays a fundamental role in modulating the reactivity of the bound flavin, but this role is at present poorly understood, in spite of numerous efforts.A few years ago, Massey et al. [l] proposed an interesting correlation between the reactivity of flavoproteins toward oxygen and toward sulfite. It was shown that a number of flavoproteins were bleached by sulfite, which reversibly formed a covalent bond with N-5 of the flavin nucleus [2]; those proteins that reacted were oxidases and formed anionic (red) semiquinones; those that did not react were dehydrogenases and formed neutral (blue) semiquinones. A tentative explanation was presented, based on these and other observations: a positive charge at the active site of oxidases would facilitate sulfite binding and stabilize the anionic semiquinone ;

Section: Circular Dichroism Measurementssupporting

confidence: 89%

Section: Circular Dichroism Measurementsmentioning

confidence: 86%

Section: Circular Dichroism Measurementsmentioning

confidence: 99%

See 1 more Smart Citation

Sulfite Binding to a Flavodehydrogenase, Cytochrome b₂ from Baker's Yeast

European Journal of Biochemistry

1978

“…Son spectre de dichroi'sme circulaire dans le visible differe considerablement de celui du cytochrome b, [27]. La stcechiomittrie du complexe avec le cytochrome c semble Bgalement tres diffkrente [28].…”

Section: Inhibition De La Cristallisation Par Le Fluorure De Ph6nylmcunclassified

Sur les deux formes moleculaires du cytochrome b2 de Saccharomyces cerevisiae

Jacq

1972

Eur J Biochem

Several authors showed that a modification of the enzyme must occur immediately before crystallization [ll, 121.We show here that in the presence of phenylmethylsulfonyl fluoride crystallization does not take place. We describe a new method for purifying the protein, using phenylmethylsulfonylfluoride a t all steps. The cytochrome b, purified in this manner shows kinetic properties identical with those described for the enzyme present in crude extracts (the so-called physiological cytochrome b, [ill), and significantly different from those of the crystalline enzyme.We further show that an essential structural difference between the two purified forms of the enzyme lies in their tetrameric structure : whereas the protomer of crystallized cytochrome b, is constituted by two polypeptide chains of unequal size (36000 and 21 000 daltons [8,9]), the protomer of physiological cytochrome 6 , is constituted by only one polypeptide chain of Mr 57000 daltons. The transformation of the latter form into the former must result from a proteolytic cleavage, which enables the less soluble, modified form to crystallize.I n the light of our results, it seems that physiological cytochrome b, is a tetramer composed of four polypeptide chains carrying one heme and one FMN each. It is tempting to postulate that each chain is folded into two globules linked by an exposed segment of polypeptide.Finally, our results cast doubt as to the significance of most results concerning cytochrome b,, which were obtained with the crystallized, artifactual form.Le cytochrome b,, ou L-lactic0 cytochrome c

“…Nevertheless, the C D spectra of the two types of flavin-free forms cannot be distinguished from those of the corresponding holo-enzymes and of the wild-type protein (this work and Iwatsubo & Risler, 1969;. It would appear, therefore, that the overall secondary structure of the &as-barrel is maintained in all these forms.…”

Section: Discussionmentioning

confidence: 68%

On the lack of coordination between protein folding and flavin insertion in Escherichia coli for flavocytochrome b₂ mutant forms Y254L and D282N

Gondry

et al. 1995

Protein Science

Wild-type flavocytochrome b2 @-lactate dehydrogenase) from Saccharomyces cerevisiae, as well as a number of its point mutants, can be expressed to a reasonable level as recombinant proteins in Escherichia coli (20-25 mg per liter culture) with a full complement of prosthetic groups. At the same expression level, active-site mutants Y254L and D282N, on the other hand, were obtained with an FMN/heme ratio significantly less than unity, which could not be raised by addition of free FMN. Evidence is provided that the flavin deficit is due to incomplete prosthetic group incorporation during biosynthesis. Flavin-free and holo-forms for both mutants could be separated on a Blue-Trisacryl M column. The far-UV CD spectra of the two forms of each mutant protein were very similar to one another and to that of the wild-type enzyme, suggesting the existence of only local conformational differences between the active holo-enzymes and the nonreconstitutable flavin-free forms. Selective proteolysis with chymotrypsin attacked the same bond for the two mutant holo-enzymes as in the wild-type one, in the proteasesensitive loop. In contrast, for the flavin-free forms of both mutants, cleavage occurred at more than a single bond.Identification of the cleaved bonds suggested that the structural differences between the mutant flavin-free and holo-forms are located mostly at the C-terminal end of the barrel, which carries the prosthetic group and the active site. Altogether, these findings suggest that the two mutations induce an alteration of the protein-folding process during biosynthesis in E. coli; as a result, the synchrony between folding and flavin insertion is lost.Finally, a preliminary kinetic characterization of the mutant holo-forms showed the K,,, value for lactate to be little affected; kc,, values fell by a factor of about 70 for the D282N mutant and of more than 500 for the Y254L mutant, compared to the wild-type enzyme.