title: Import of cytochrome c into mitochondria. Cytochrome c heme lyase.
abstract: The import of cytochrome c into mitochondria can be resolved into a number of discrete steps. Here we report on the covalent attachment of heme to apocytochrome c by the enzyme cytochrome c heme lyase in mitochondria from Neurospora crassa. A new method was developed to measure directly the linkage of heme to apocytochrome c. This method is independent of conformational changes in the protein accompanying heme attachment. Tryptic peptides of [35S]cysteine-labelled apocytochrome c, and of enzymatically formed holocytochrome c, were resolved by reverse-phase HPLC. The cysteine-containing peptide to which heme was attached eluted later than the corresponding peptide from apocytochrome c and could be quantified by counting 35S radioactivity as a measure of holocytochrome c formation. Using this procedure, the covalent attachment of heme to apocytochrome c, which is dependent on the enzyme cytochrome c heme lyase, could be measured. Activity required heme (as hemin) and could be reversibly inhibited by the analogue deuterohemin. Holocytochrome c formation was stimulated 5--10-fold by NADH greater than NADPH greater than glutathione and was independent of a potential across the inner mitochondrial membrane. NADH was not required for the binding of apocytochrome c to mitochondria and was not involved in the reduction of the cysteine thiols prior to heme attachment. Holocytochrome c formation was also dependent on a cytosolic factor that was necessary for the heme attaching step of cytochrome c import. The factor was a heat-stable, protease-insensitive, low-molecular-mass component of unknown function. Cytochrome c heme lyase appeared to be a soluble protein located in the mitochondrial intermembrane space and was distinct from the previously identified apocytochrome c binding protein having a similar location. A model is presented in which the covalent attachment of heme by cytochrome c heme lyase also plays an essential role in the import pathway of cytochrome c.
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000032:analysis; D003574:Cytochrome c Group / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006418:Heme / Q000378:metabolism; D006427:Hemin / Q000031:analogs & derivatives / Q000494:pharmacology; D008190:Lyases / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009492:Neurospora crassa / Q000201:enzymology / Q000648:ultrastructure; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D012156:Reticulocytes / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D003574:Cytochrome c Group; D010446:Peptide Fragments; D009243:NAD; C011219:deuterohemin; D006418:Heme; D009249:NADP; D006427:Hemin; D014357:Trypsin; D008190:Lyases; C031581:cytochrome C synthetase; D003545:Cysteine; D005033:Ethylmaleimide