Sur les deux formes moleculaires du cytochrome b2 de Saccharomyces cerevisiae

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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 ;

“…Both enzymes act on L-lactate, with, it is true, rather different K, values: 0.4mM and 22 mM respectively [9,26]. Both are inhibited by D-lactate, and by oxalate [27].…”

Section: Discussionmentioning

confidence: 99%

“…Except where stated, all experiments were carried out with the intact form of cytochrome 62 [8]; the nicked form has the same quaternary structure, but somewhat altered catalytic properties [8,9].…”

mentioning

confidence: 99%

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

Lederer

1978

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“…Very recently, this ((two chains per monomer" structure for crystallized cytochrome b, was shown to result from a proteolytic cleavage [25,26]. Actually a "one chain per monomer" structure is found in the cytochrome b, of Hansenula anornula, and in the non-crystallized preparations of cytochrome b, of Saccharomyces cerevisiae.…”

Section: Discussionmentioning

confidence: 99%

Subunit Structure of L‐Lactate Dehydrogenase (Cytochrome b₂) of Saccharomyces cerevisiae

Mével-Ninio¹

1972

The molecular weights of yeast L‐lactate dehydrogenase (cytochrome b2) and of its polypeptide chains have been measured by ultracentrifugation. The intact protein was found to have a molecular weight of 240000 ± 10000 by sedimentation equilibrium measurements according to the Yphantis high‐speed method. The solution obtained by dissolving the protein in 6 M guanidine (with added 2‐mercaptoethanol) behaved as if it were monodisperse, giving Mr= 31300 ± 2000, by approach‐to‐equilibrium measurements. This result indicates that the “58 600 unit” associated to one set of prosthetic groups, heme and flavin, is made up of two chains, and thus the whole molecule contains a total of eight chains, identical or not. However, as the viscosity of the medium was reduced (using 2 M guanidine) the paucidispersity of the cytochrome b2 solution was revealed. The mixture was shown to consist of two kinds of chains distinct in molecular weight. From these experiments the molecular weight of the light chain was found to be 22600 ± 1500. After partial separation of the two chains by gel filtration through Sephadex G‐100, the molecular weight of the heavy chain was estimated as 39700 ± 3000. Preliminary experiments indicate that the heavy component can bind quantitatively the heme prosthetic group. The FMN appears to be bound to the light component.

“…The two forms present a number of catalytic as well as physicochemical differences [2] : the intact enzyme, in particular, shows a more than two-fold higher specific activity, smaller K, and K values for substrate and inhibitors, and is inhibited by excess substrate. Moreover, it is still soluble at the low salt concentrations which are used to crystallize the cleaved enzyme.…”

mentioning

confidence: 99%

“…It is known under two molecular forms. When purified in the presence of phenylmethylsulfonylfluoride according to Jacq and Lederer [l], it is a tetramer of four presumably identical subunits with apparent molecular weight 57000 [1,2]. When the enzyme is purified by the classical procedure of Appleby and Morton [3], where crystallization from a partly fractionated autolysate is the main purification step, dodecylsulfateacrylamide gel electrophoresis shows two bands with molecular weight 33 -36000 and 21 000 (subunits CI and p respectively) [4].…”

mentioning

confidence: 99%

Controlled Proteolysis of Flavocytochrome b₂

Pompon¹,

Lederer²

1976

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It is known that each subunit of the tetrameric flavocytochrome b2 can be cleaved by yeast proteases to fragments of molecular weight 33‐36000 and 21000, with some modification of catalytic properties, but without destruction of the oligomeric state of the protein. We report here experimental conditions which enabled us to simulate this specific cleavage in a controlled fashion with chymotrypsin and subtilisin. With trypsin and papain, on the other hand, it was not found possible to stop the digestion in such a way as to obtain a homogeneous still active product. A characterization of the enzymatic forms obtained by digestion with chymotrypsin and subtilisin at 0 °C shows that modification of enzymatic and solubility properties occurs in a stepwise fashion. It is also concluded that cleavage by yeast proteases is accompanied by loss of 10 to 25 residues. At 37 °C, chymotrypsin digestion yields a heme‐binding core of molecular weight 15000, larger than the already characterized tryptic heme‐binding core by about 40 residues. Although the latter is known to be very similar to trypsin‐solubilized cytochrome b5, the lack of aggregation of the former in aqueous solution, its amino acid composition and circular dichroism spectra do not point to a similarity of its additional peptide segment with the hydrophobic tail of detergent‐solubilized cytochrome b5.