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 > NADPH > 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.

MOM19, an import receptor for mitochondrial precursor proteins

“…Following reisolation, mitochondria were incubated with 30 pl of reticulocyte lysate containing [35S]cysteinelabeled apocytochrome c in SEM buffer containing hemin (3 pM) and sodium dithionite (1 mg/ml) for 10 min at 25%. Levels of holocytochrome c formed were determined following immunoprecip itation, digestion of immunocomplexes with trypsin, and subjection to reverse-phase HPLC (Nicholson et al, 1967). Isolated mitochondria were pretreated with trypsin (15 tug/ml) as described (Pfanner and Neupert, 1967a).…”

Synthesis and Assembly of Mitochondrial Proteins

“…In this case, however, the stimulatory component is a low molecular weight, heat-stable factor which is not sensitive to proteases. In yeast, the cofactor can be substituted by an NADPH-regenerating System (Taniuchi et aL, 1982), but in N. crassa the cofactor appears to serve some other function (Nicholson et aL, 1987). It is not involved in the binding of apocytochrome c to mitochondria, but is necessary for enzymatic attachment of heme and subsequent translocation across the outer membrane.…”

Covalent cofactor binding to flavoenzymes requires specific effectors

“…Binding of heme to apocytochrome c requires, besides the enzyme cytochrom-c-heme lyase, the reduced nucleotide NADH as cofactor [3] and in Neurospora crassa mitochondria an additional cytosolic factor of as yet unknown nature [18]. The reaction in which biotin is bound to a lysine residue in carboxylases is catalyzed by biotin-holoenzyme synthetase.…”