Identification of porphyrin present in apo-cytochrome c oxidase of copper-deficient yeast cells

• Published in: Biochimica et biophysica acta Vol. 633; no. 2; pp. 211 - 227
• Main Authors: Keyhani, Ezzatollah, Keyhani, Jacqueline
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.1980
• Subjects: Aminolevulinic Acid - metabolism; Apoenzymes - metabolism; Apoproteins - metabolism; Chromatography, Thin Layer; Copper - deficiency; Cytochrome c oxidase; Electron Transport Complex IV - immunology; Electron Transport Complex IV - metabolism; Electrophoresis, Polyacrylamide Gel; Heme - analogs & derivatives; Heme - metabolism; Mitochondria - analysis; Molecular Weight; Porphyrin a; Porphyrins - biosynthesis; Porphyrins - isolation & purification; Saccharomyces cerevisiae - enzymology; Spectrophotometry; Yeast; Cytochrome c oxidase; Yeast; Copper deficiency; Porphyrin a
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(80)90407-9

Heme a was not detected either in mitochondria isolated from copper-deficient yeast or in the intact cells. Nevertheless, the intracellular concentration of free porphyrins indicated that the pathway of porphyrin and heme synthesis was not impaired in copper-deficient cells. The immunoprecipitated apo-oxidase from copper-deficient cells revealed an absorption spectrum with maxima at 645, 592, 559, 519 and 423 nm, similar to that of purified porphyrin a. When solubilized mitochondria from [ 3H]leucine and δ-amino[ 14C]levulinic acid-labeled copper-deficient yeast cells were incubated with rabbit antiserum against cytochrome c oxidase, a precipitate was obtained. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of this immunoprecipitate showed [ 3H]leucine associated with six bands and δ-amino[ 14C]levulinic acid resolved in a single band. HCl fractionation of copper-deficient mitochondria labeled with δ-amino[ 14C]levulinic acid showed a high specific radioactivity in the fraction extracted by 20% HCl, a solvent which extracts porphyrin a. Thinlayer chromatography of the radioactivity found in 20% HCl showed an R F value identical to that of purified porphyrin a. When δ-amino[ 3H]levulinic acid-labeled, copper-deficient yeast cells are grown in copper-supplemented medium, the porphyrin a accumulated in copper-deficient cells wa converted into heme a, and this conversion was prevented by cycloheximidine. These observations suggest that porphyrin a is present in the apo-oxidase of copper-deficient cells, but that the conversion to heme a does not occur. This conversion reaction appears to be a point in the biosynthetic pathway of cytochrome c oxidase which is blocked by copper deficieny.