Maturation of the unusual single-cysteine (XXXCH) mitochondrial c-type cytochromes found in trypanosomatids must occur through a novel biogenesis pathway

Abstract

The c-type cytochromes are characterized by the covalent attachment of haem to the polypeptide via thioether bonds formed from haem vinyl groups and, normally, the thiols of two cysteines in a CXXCH motif. Intriguingly, the mitochondrial cytochromes c and c1 from two euglenids and the Trypanosomatidae contain only a single cysteine within the haem-binding motif (XXXCH). There are three known distinct pathways by which c-type cytochromes are matured post-translationally in different organisms. The absence of genes encoding any of these c-type cytochrome biogenesis machineries is established here by analysis of six trypanosomatid genomes, and correlates with the presence of single-cysteine cytochromes c and c1. In contrast, we have identified a comprehensive catalogue of proteins required for a typical mitochondrial oxidative phosphorylation apparatus. Neither spontaneous nor catalysed maturation of the single-cysteine Trypanosoma brucei cytochrome c occurred in Escherichia coli. However, a CXXCH variant was matured by the E. coli cytochrome c maturation machinery, confirming the proposed requirement of the latter for two cysteines in the haem-binding motif and indicating that T. brucei cytochrome c can accommodate a second cysteine in a CXXCH motif. The single-cysteine haem attachment conserved in cytochromes c and c1 of the trypanosomatids is suggested to be related to their cytochrome c maturation machinery, and the environment in the mitochondrial intermembrane space. Our genomic and biochemical studies provide very persuasive evidence that the trypanosomatid mitochondrial cytochromes c are matured by a novel biogenesis system.

Figure 1. Absorption spectra of the recombinant CXXCH variant of Trypanosoma brucei cytochrome c and of wild-type Crithidia fasciculata cytochrome c

The CXXCH variant of T. brucei cytochrome c was purified from periplasmic extracts of E. coli cells co-transformed with the plasmid for the E. coli Ccm system. The spectrum (solid line) was recorded at 25 °C with the protein in 50 mM Tris/HCl, pH 7.5, following addition of a few grains of disodium dithionite. Wild-type C. fasciculata cytochrome c was purified from the mitochondria of a chanomastigote culture; the spectrum (broken line) was recorded under the same conditions as above, except at pH 8.0. In the absence of accurate molar absorption coefficients, these spectra have been normalized by the Soret band intensity. Inset: reduced pyridine haemochrome spectra of the CXXCH variant of T. brucei cytochrome c purified from the periplasm of E. coli (solid line) and of C. fasciculata cytochrome c (broken line). The final concentrations of NaOH and pyridine were 0.2 M and 30% (v/v) respectively; spectra were normalized by the α-band intensity.