The biosynthesis of heme O and heme A is not regulated by copper.

Heme A is an obligatory cofactor in all eukaryotic and many prokaryotic cytochrome c oxidase (CcO) enzymes. Despite its obvious importance to CcO and the electron transport pathway, essentially nothing is known concerning the regulation of heme A. Because CcO is the only natural target for heme A and copper is also required for CcO activity, it was postulated that copper might regulate heme A homeostasis. Work reported previously demonstrated that there is often a strong connection between copper and iron homeostasis in general, and circumstantial evidence pointed to a possible specific link between copper and heme A. To address this question, we conducted experiments to determine rigorously whether copper plays a role in heme A homeostasis. The two enzymes responsible for the conversion of heme B to heme A, heme O synthase (HOS) and heme A synthase (HAS), were separately genomically epitope-tagged in Saccharomyces cerevisiae, and their expression under various copper conditions was quantified by Western blot analysis. These results demonstrated that the sum of transcription, translation, and stability of HOS and HAS were independent of copper. Additionally, the effects of intracellular copper concentrations on the activity of HOS and HAS from Bacillus subtilis (expressed in Escherichia coli) and Rhodobacter sphaeroides were examined by analysis of cellular heme extracts. No trends with respect to intracellular copper were observed. In combination, our results demonstrate that intracellular copper levels do not affect the transcription, translation, stability, or activity of either HOS or HAS.