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

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 c1 (CC1HL). 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 c2 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.

► We test the substrate specificity of Saccharomyces cerevisiae cytochrome c heme lyase (CCHL).
► CCHL can only ligate heme b groups to apocytochromes c of eukaryotic origin.
► The amino-terminal region of eukaryotic apocytochromes c is critical for CCHL activity.
► We convert a prokaryotic apocytochrome c into an efficient CCHL substrate with two mutations.
► We define a consensus sequence for holocytochrome c formation by CCHL.