Investigating the Catalytic Mechanism of Yeast Cytochrome c Heme Lyase (CCHL) Through Mutation of Highly Conserved Amino Acid Residues

Abstract

Cytochrome c is a vital component in energy transduction pathways, as well as in the process of programmed cell death (apoptosis). As a functional holoform, cytochrome c contains at least one covalently attached heme cofactor. In fungi, metazoans, and some protozoa, heme attachment is catalyzed by the enzyme cytochrome c heme lyase (CCHL), which is also known as holocytochrome c synthase (HCCS). In humans, genetic mutations leading to nonfunctional CCHL are lethal to males and cause microphthalmia with linear skin defects syndrome (MLS) in females. Despite this association with human disease, little is known about the mechanistic and structural properties of CCHL. Here, we used yeast CCHL to carry out site‐directed mutagenesis to evaluate the role of highly conserved residues adjacent to histidine at position 128 (H128), which has been found to be critical for catalytic activity. Generated mutants were co‐expressed with yeast apocytochrome c in an Escherichia coli system, in which successful holocytochrome c formation in vivo is indicated by the development of red colored cells (red phenotype). Cells coexpressing yeast apocytochrome c and CCHL mutants H128N and H128C lost the ability to ligate heme to apoprotein substrates (white phenotype). This ability was retained at varying levels, however, in M124C, V125C, Q126C, 127C, F130C, L131C, and N132C mutants. Absorbance spectroscopy confirmed these findings, revealing characteristic spectra for holocytochrome c in the lysates of red (but not white) colored cells. In some mutants, it has also been observed that the redox behavior of produced holocytochrome c differs from that in wildtype. These mutants are being investigated for potential altered enzyme‐substrate interactions.