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.

The cytokine TNF-alpha plays a major role in inflammatory and immunological reactions of canine skin. With respect to a possible therapeutic modulation, we investigated the role of the transcription factor NF-kappa B and the involvement of reactive oxygen species (ROS) in the TNF-alpha signalling pathway in cultured canine keratinocytes. TNF-alpha treatment resulted in activation of NF-kappa B which was partially inhibited by the antioxidant alpha-lipoic acid. Using the cytochrome c reduction test no superoxide production could be detected in the supernatant of TNF-alpha stimulated keratinocytes. However, TNF-alpha dependent intracellular hydrogen peroxide production was demonstrated spectroscopically. With electron energy loss spectroscopy (EELS) significant hydrogen peroxide formation was detected in the mitochondria, the endoplasmic reticulum, the cytosol and partially on the plasma membrane of the keratinocytes. Hence, ROS possibly play an important role in the TNF-alpha signalling pathway leading to NF-kappa B activation in canine skin. An adjunctive therapy with natural potent antioxidants modulating NF-kappa B overactivation in canine cutaneous inflammation may be of therapeutic benefit.

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