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Mammalian cytochrome c (Cyt c) has two primary functions: transfer of electrons from the bc1 complex to cytochrome c oxidase (COX) as part of the mitochondrial electron transport chain (ETC), and participation in type II apoptosis. Several studies have indicated that components of the ETC can be phosphorylated, and we have recently shown that the Cyt c electron acceptor COX is phosphorylated on Tyr-304 of subunit I in liver upon activation of the cAMP-dependent pathway, leading to strong enzyme inhibition. However, covalent modification of Cyt c through phosphorylation has not yet been reported. We have isolated Cyt c from cow heart under conditions that preserve the physiological in vivo phosphorylation status. Western analysis with an anti-phosphotyrosine antibody indicated tyrosine phosphorylation. The site of phosphorylation was definitively assigned by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS) to Tyr-97, one of the four tyrosine residues present in Cyt c. The phosphorylated tyrosine is part of a motif that contains five residues identical to the tyrosine phosphorylation site in COX subunit I. Spectral analysis revealed that the characteristic 695 nm absorption band is shifted to 687 nm and reversed after treatment with alkaline phosphatase. This band results from the Met-80-heme iron bond, and its shift might indicate changes in the catalytic heme crevice. In vivo phosphorylated Cyt c shows enhanced sigmoidal kinetics with COX, and half-maximal turnover is observed at a Cyt c substrate concentration of 5.5 microM compared to 2.5 microM for alkaline phosphatase-treated Cyt c. Possible consequences of Tyr-97 phosphorylation with respect to cardiolipin binding and of location of Tyr-97 in close proximity to Lys-7, a crucial residue for interaction with Apaf-1 during apoptosis, are discussed.

Mammalian cytochrome c oxidase (COX) is a 13-subunit polypeptide complex that contains 10 subunits coded by the nucleus and 3 by the mitochondria. The nuclear-encoded subunits, though of unknown function, are presumed to play a regulatory role. Three of these (subunits VIa, VIIa, and VIII) generally exist in one of two isoforms--a constitutive (L) isoform or a skeletal muscle/heart-specific (H) isoform. To study the regulation, and possibly function, of these isoforms, we have begun characterizing the genes. In this paper we describe the isolation and characterization of the gene for the bovine COX VIIa-H isoform. The gene consists of four exons spanning 1.58 kb and is associated with a CpG island. There are no canonical TATA or CCAAT boxes immediately upstream of the transcription start site. Putative DNA sequence elements associated with respiratory function, muscle gene activation, and housekeeping function are present both in the upstream regions and within introns.