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

Abstract

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.

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

“…8) [9,43,44]. Preliminary reconstitution experiments with the partly purified m and ,!j subunits of cleaved cytochrome b, also suggested that haem binding occurred preferentially on the m subunit, whereas the evidence concerning the FMN binding site looked rather weak [27]. However, even before conclusive experimental evidence becomes available, it now seems quite probable that it is located on the segment corresponding to the light chain of the cleaved enzyme for the reasons that follow.…”

Three-dimensional structure of flavocytochrome b2 from baker's yeast at 3.0-A resolution.

“…Flavocytochrome b2 [L-lactate:ferricytochrome c oxidoreductase, EC 1.1.2.3] from baker's yeast (Saccharomyces cerevisiae) is a tetrameric enzyme of Mr 230,000 (1) and is located in the intermembrane space of mitochondria (2). Each subunit of Mr 57,500 (3) contains one heme and one FMN, both bound noncovalently (4).…”

Study of the Hansenula anomala yeast flavocytochrome‐b2–cytochrome‐c complex 1. Characterization of fluorescent Zn(II)‐substituted cytochrome c

“…Yeast L-lactate :cytochrome c oxidoreductase, commonly referred to as flavocytochrome b,, consists of a tetramer [3,4]. Each protomer contains one flavin mononucleotide and one protoheme [5] coordinated by two histidine residues [6].…”