Primary structure of flavocytochrome <i>b</i><sub>2</sub> from baker's yeast

The yeast L(+)‐lactate cytochrome c oxidoreductase or cytochrome b2 is a component of the mitochondrial intermembrane space. The protein is encoded by the nuclear genome, synthesized as a larger precursor in the cytoplasmic compartment, and then proteolytically processed to its mature form during its import into the mitochondria. The structural gene for yeast cytochrome b2 has been cloned. The complete nucleotide sequence of the gene with its 5′ and 3′ flanking regions was determined. The deduced primary structure of the cytochrome b2 precursor reveals an unusually long amino terminal extension of 80 amino acids. A variety of potentially significant sequences were identified in the region flanking the structural portion of the gene. Transcript mapping with both S1 nuclease and primer extension methods reveals that the site of RNA synthesis is 56‐66 bp downstream from a putative TATA box. By Northern blot analysis and gene disruption, it is shown that there is only a single copy of the cytochrome b2 gene per haploid yeast nucleus. The cloned cytochrome b2 gene was used to probe specific mRNA levels and demonstrate that cytochrome b2 expression is transcriptionally repressed by glucose and induced by lactate. The inactivation of the chromosomal cytochrome b2 gene by integrative transformation led to a deficiency in L(+)‐lactate dehydrogenase activity and consequently to the inability to use L(+)‐lactate as a sole source of carbon. This is the first reported mutation affecting the structural gene of cytochrome b2.

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“…The deduced sequence of mature cytochrome b2 confirms the partial primary structure presented previously (Guiard et al, 1974;Ghrir et al, 1984).…”

Section: Bguiardsupporting

confidence: 88%

“…wt. of 56 569 and 511 amino acid residues, and confirms the partial amino acid sequence presented previously (Guiard et al, 1974;Ghrir et al, 1984). Mapping the cytochrome b2 transcript The open reading frame described above delineated the coding region of the pre-cytochrome b2.…”

Section: Isolation Of the Gene Encoding Cytochrome B2supporting

confidence: 86%

Structure, expression and regulation of a nuclear gene encoding a mitochondrial protein: the yeast L(+)-lactate cytochrome c oxidoreductase (cytochrome b2).

Guiard¹

1985

The EMBO Journal

180

150

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“…Incubation with [14C]phenyglyoxal led to the results presented in Figure 4. Flavocytochrome b2 contains 18 arginine residues per subunit and about 44 lysine residues (Lederer & Simon, 1971;Ghrir et al, 1984; S. Coctial, A. M. Becam, and F. Lederer, unpublished experiments). Amino acid analyses after hydrolysis gave no reliable indication for arginine or lysine loss.…”

Section: Resultsmentioning

confidence: 94%

Inactivation of flavocytochrome b2 from bakers' yeast with phenylglyoxal

Ghrir¹,

Lederer²

1984

Biochemistry

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Extreme pK_a displacements at the active sites of FMN‐dependent α‐hydroxy acid‐oxidizing enzymes

Lederer

1992

Protein Science

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Flavocytochrome b2 (or L-lactate dehydrogenase) from baker's yeast is thought to operate by the initial formation of a carbanion, as do the evolutionarily related alpha-hydroxy acid-oxidizing FMN-dependent oxidases. Previous work has shown that, in the active site of the unligated reduced flavocytochrome b2, the group that has captured the substrate alpha-proton has a high pKapp, calculated to lie around 15 through the use of Eigen's equation. A detailed inspection of the now known three-dimensional structure of the enzyme leads to the conclusion that the high pKa belongs to His 373, an active site group that plays the role of general base in the forward reaction and of general acid in the reverse direction. Moreover, consideration of the kinetics of proton transfer during the catalytic cycle suggests that the pKa of the reduced FMN N5 position should be lowered by several pH units compared to its pKa of 20 or more when free. The features of the three-dimensional structure possibly responsible for these pK shifts are analyzed; they are proposed to consist of a network of hydrogen bonds with the solvent and of a mutual electrostatic stabilization of anionic reduced flavin and the imidazolium ion. Finally, it is suggested that similar pK shifts affect the active sites of the alpha-hydroxy acid-oxidizing flavooxidases, which are homologous to flavocytochrome b2. The functional significance of these pK shifts in terms of catalysis and semiquinone stabilization is discussed.

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Primary structure of flavocytochrome b₂ from baker's yeast

Cited by 9 publications

References 72 publications

Structure, expression and regulation of a nuclear gene encoding a mitochondrial protein: the yeast L(+)-lactate cytochrome c oxidoreductase (cytochrome b2).

Structure, expression and regulation of a nuclear gene encoding a mitochondrial protein: the yeast L(+)-lactate cytochrome c oxidoreductase (cytochrome b2).

Inactivation of flavocytochrome b2 from bakers' yeast with phenylglyoxal

Extreme pK_a displacements at the active sites of FMN‐dependent α‐hydroxy acid‐oxidizing enzymes

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Primary structure of flavocytochrome b2 from baker's yeast

Cited by 9 publications

References 72 publications

Structure, expression and regulation of a nuclear gene encoding a mitochondrial protein: the yeast L(+)-lactate cytochrome c oxidoreductase (cytochrome b2).

Structure, expression and regulation of a nuclear gene encoding a mitochondrial protein: the yeast L(+)-lactate cytochrome c oxidoreductase (cytochrome b2).

Inactivation of flavocytochrome b2 from bakers' yeast with phenylglyoxal

Extreme pKa displacements at the active sites of FMN‐dependent α‐hydroxy acid‐oxidizing enzymes

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Product

Resources

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Primary structure of flavocytochrome b₂ from baker's yeast

Extreme pK_a displacements at the active sites of FMN‐dependent α‐hydroxy acid‐oxidizing enzymes