Engineering a prokaryotic apocytochrome c as an efficient substrate for Saccharomyces cerevisiae cytochrome c heme...

Cytochromes c are heme proteins that require multiple maturation components, such as heme lyases, for cofactor incorporation. Saccharomyces cerevisiae has two heme lyases that are specific for apocytochromes c (CCHL) or c(1) (CC(1)HL). CCHL can covalently attach heme b groups to apocytochrome c substrates of eukaryotic but not prokaryotic origin. Besides their conserved Cys-Xxx-Xxx-Cys-His heme-binding motifs, the amino-terminal regions of apocytochrome c substrates appear to be important for CCHL function. In this study, we show for the first time that only two amino acid changes in the amino-terminal region of the non-CCHL substrate apocytochrome c(2) from Rhodobacter capsulatus are necessary and sufficient for efficient holocytochrome c formation by CCHL. This finding led us to propose a consensus sequence located at the amino-terminus of apocytochromes c, and critical for substrate recognition and heme ligation by CCHL.