Amino‐acid sequence of the cytochrome‐b₅‐like heme‐binding domain from Hansenula anomala flavocytochrome b₂

Abstract

Flavocytochrome b2 (L-lactate dehydrogenase) from baker's yeast is composed of two structural and functional domains. Its first 100 residues constitute the heme-binding core, which is homologous to cytochrome b5 [B. Guiard, O. Groudinsky & F. Lederer (1974) Proc. Natl Acad. Sci. USA 71, 2539-2543]. We report here the amino acid sequence of the heme-binding domain isolated by tryptic proteolysis of Hansenula anomala flavocytochrome b2. The sequence was established by automated degradation of the whole fragment and of peptides obtained by CNBr cleavage at the unique tryptophan and by proteolysis with thermolysin and endoproteinase Lys C. As isolated, the domain consists of 84 residues without any sulfur amino acids. It shows 49 identities with the heme-binding domain from Saccharomyces cerevisiae and 28 with beef microsomal cytochrome b5. Using the recently published three-dimensional structure of S. cerevisiae flavocytochrome b2 [Z-x. Xia, N. Shamala, P. H. Bethge, L. W. Lim, H. D. Bellamy, N. H. Xuong, F. Lederer and F. S. Mathews (1987) Proc. Natl Acad. Sci. USA 84, 2629-2633], it can be seen that there are only positively charged side chains close to the accessible heme edge, the only negative charges in that area being those of the heme propionates. The implications of this result are discussed in the light of Salemme's model for the cytochrome b5/cytochrome c complex [F. R. Salemme (1976) J. Mol. Biol. 102, 563-568].

Structural basis for the kinetic differences between flavocytochromes b2 from the yeasts Hansenula anomala and Saccharomyces cerevisiae

“…The predicted amino acid sequence completely confirms the published sequence of the isolated cytochrome b2'core' domain [17], and extends the N-terminal domain by six residues, but two discrepancies were found when our predicted sequence was compared with the protein sequence of part of the C-terminal flavin domain [16]. We found residue 352 to be glutamine rather than glutamic acid and residue 365 was leucine as opposed to isoleucine.…”

“…All the active-site residues thought to be directly involved in catalysis (His-373, Tyr-254, Arg-376, Lys-349, Tyr-143 and Asp-282) [5] and several others identified as being important for flavin and haem binding are identical in the two species. As discussed by Haumont et al [17], the residues buried within the N-terminal haem domain are well conserved and it can be assumed that these fold very similarly. The C-terminal flavin-containing domain of S. cerevisiae flavocytochrome b2 contains an a8/38 barrel [2].…”

Expression in Escherichia coli of the flavin and the haem domains of Hansenula anomala flavocytochrome b2 (flavodehydrogenase and b2 core) and characterization of the recombinant proteins¶

“…The tryptophan content of the molecule, determined by emission fluorescence spectroscopy (see the Materials and methods section), is 2.5 tryptophan residues per haem; two tryptophan residues are expected on the basis of the amplified sequence. It must be recalled that p-core contains one (Haumont et al, 1987) In order to probe the structure of the chromophore in the purified haemoprotein by a method more sensitive than optical spectroscopy, we have obtained the r.R. spectra of r-core in the high-, mediumand low-frequency regions in order to obtain a comparison with the r.R.…”

Amperometric Biosensors for Lactate, Alcohols, and Glycerol Assays in Clinical Diagnostics

“…Besides LDH and LOX, another enzyme is known for participating in the lactic acid metabolism in yeasts, namely L-lactate-cytochrome c oxidoreductase (EC 1.1.2.3; flavocytochrome b 2 , FC b 2 ) (Brooks, 2002), which catalyses the electron transfer from L-lactate to cytochrome c in yeast mitochondria. The protein can be isolated from Saccharomyces cerevisiae and Hansenula anomala (Labeyrie et al, 1978;Haumont et al, 1987;Silvestrini et al, 1993) as a tetramer with four identical subunits, each consisting of FMN-and heme-binding domains. FC b 2 has absolute specificity for L-lactate, moreover, it functions in vitro without regard to the nature of electron acceptors which makes this enzyme very promising for analytical biotechnology.…”