Identification of porphyrin present in apo-cytochrome C oxidase of copper-deficient yeast cells.
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 delta-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 delta-amino[14C]levulinic acid resolved in a single band. HCl fractionation of copper-deficient mitochondria labeled with delta amino[14C]levulinic acid showed a high specific radioactivity in the fraction extracted by 20% HCl, a solvent which extracts porphyrin a. Thin-layer chromatography of the radioactivity found in 20% HCl showed an RF value identical to that of purified porphyrin a. When delta-amino[3H]levulinic acid-labeled, copper-deficient yeast cells are grown in copper-supplemented medium, the porphyrin a accumulated in copper-deficient cells was 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 deficiency.
D000622:Aminolevulinic Acid / Q000378:metabolism; D001051:Apoenzymes / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D003300:Copper / Q000172:deficiency*; D003576:Electron Transport Complex IV / Q000276:immunology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000031:analogs & derivatives* / Q000378:metabolism; D008928:Mitochondria / Q000032:analysis; D008970:Molecular Weight; D011166:Porphyrins / Q000096:biosynthesis / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013053:Spectrophotometry
D001051:Apoenzymes; D001059:Apoproteins; D011166:Porphyrins; C027728:heme a; C028285:porphyrin a; D006418:Heme; D003300:Copper; D000622:Aminolevulinic Acid; D003576:Electron Transport Complex IV