Cytochrome b₂ from Bakers' Yeast (L‐Lactate Dehydrogenase)

Abstract

Bakers' yeast cytochrome b, [L-( +)-lactate dehydrogenase or L-( +)-lactate cytochrome c oxidoreductase], usually purified by crystallisation according to Appleby and Morton, was shown to be a degraded form of the enzyme, arising by a proteolytic modification of the species formed in vivo. We present here an improved purification procedure and some properties of the intact enzyme.This intact cytochrome b, is shown, by cross-linking and by gel filtration studies, to be in a tetrameric form with a molecular weight of about 230000. The four identical subunits are composed of a single polypeptide chain associated with one haem and one flavin (FMN). The amino-acid composition is presented. Alanine and glutamic acid are shown to be respectively C-and Nterminal residues.A comparison of these results with those concerning the degraded form suggests that there is little loss of peptide material and that the gross quaternary structure of the protein is not modified by the proteolytic process. However circular dichroism studies show that the haem environment is altered.The evidence leading to propose a biglobular structure for the enzyme is discussed. We report here an improved purification procedure for the intact cytochrome b,. The quaternary structure of this protein obtained under a stable form is also characterized and compared with the known structure of the cleaved enzyme. I n our previous paper [6] we used the terms physiological [5] and crystallized to distinguish the two forms of the protein, but we shall now only use the terms intact and cleaved, which are immediately understandable.A preliminary report of this work has been presented before [7]. MATERIALS AND METHODS MaterialsFresh baker's yeast was obtained from Springer (Maisons Alfort France) and was lyophilized just before extraction. DEAE-cellulose (DE 32) was purchased from Whatman, 1-ethyl-3 (3-dimethyl aminopropy1)-carbodiimide dihydrochloride from Pierce, 1,g-hexanediamine dihydrochloride and cyanogen bromide from Eastman. Phenylmethylsulfonyl fluoride and other chemicals came from Merck and were of the highest degree of purity available.

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

“…sulfite complex was determined in the visible range and is compared in Fig. 7 to the oxidized and reduced spectra, already determined by Jacq and Lederer [8]. Whereas a distinct signal had been observed for the cleaved enzyme Soret band [19,20], Jacq and Lederer [8] found no optical activity associated with the intact enzyme in the Soret region.…”

“…Clearly, circular dichroism detects no influence of the flavin redox state on the interaction of the oxidized heme with its surrounding aromatic groups [21]. As completely oxidized and completely reduced cytochrome b2 show practically no difference in the ellipticity of the peptide chromophores [8,19,20], the corresponding region was not examined in this work.…”

“…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].…”

Controlled Proteolysis of Flavocytochrome b2

“…The enzyme is still a tetramer [5,6]. Chemical studies have led to the conclusion that, in the autolysis mixture, yeast proteases remove seven residues from the amino-terminal of each subunit and cleave it in a region about two-thirds down the chain [1,7]. It had not been possible at the time (EC 3.4.21.14); papain (EC 3.4.22.2). to define exactly how much peptide material was removed in the process.…”

“…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].…”