Studies on the biosynthesis of cytochrome c.
A soluble cytochrome was isolated and purified from the slime mould Physarum polycephalum and identified as cytochrome c by room-temperature and low-temperature (77 degrees K) difference spectroscopy. A close similarity between P. polycephalum and mammalian cytochromes c was suggested by a comparison of the initial rates of oxidation of both proteins by mammalian mitochondria. This similarity was further emphasized by redox titrations and gel-electrophoretic studies which indicated that P. polycephalum cytochrome c has an oxidation-reduction midpoint potential of +257mV at pH7.0 and a molecular weight of 12500+/-1500 (mean+/-maximum deviation for a set of six measurements). P. polycephalum exhibits an absolute requirement for protohaemin for growth. The (59)Fe-labelled haemin was prepared by chemical synthesis from protoporphyrin. The purified product had a specific radioactivity of 0.8+/-0.02muCi/mol. Growth of P. polycephalum in the presence of [(59)Fe]haemin resulted in the incorporation of (59)Fe into the plasmodial cytochrome c. The specific radioactivity of the cytochrome c haem was 0.36+/-0.02muCi/mol. The high specific radioactivity of the cytochrome haem indicates that synthesis of the holoenzyme must proceed by direct attachment of haem to the apoprotein rather than by the intermediate formation of a protoporphyrinogen-apoprotein complex. The observed decrease in the specific radioactivity of the haem group is attributed to exchange of the (59)Fe with unlabelled iron in the plasmodia either before or during attachment of the haem group to the apoprotein.
D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002854:Chromatography, Paper; D003574:Cytochrome c Group / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000378:metabolism; D006736:Horses; D007503:Iron Isotopes; D008930:Mitochondria, Liver / Q000378:metabolism; D008970:Molecular Weight; D009206:Myocardium; D009235:Myxomycetes / Q000201:enzymology*; D010084:Oxidation-Reduction; D011485:Protein Binding; D051381:Rats; D013045:Species Specificity; D013053:Spectrophotometry; D013696:Temperature
D001059:Apoproteins; D003574:Cytochrome c Group; D007503:Iron Isotopes; D006418:Heme