Thiol redox requirements and substrate specificities of recombinant cytochrome c assembly systems II and III

Abstract

The reconstitution of biosynthetic pathways from heterologous hosts can help define the minimal genetic requirements for pathway function and facilitate detailed mechanistic studies. Each of the three pathways for the assembly of cytochrome c in nature (called systems I, II, and III) has been shown to function recombinantly in Escherichia coli, covalently attaching heme to the cysteine residues of a CXXCH motif of a c-type cytochrome. However, recombinant systems I (CcmABCDEFGH) and II (CcsBA) function in the E. coli periplasm, while recombinant system III (CCHL) attaches heme to its cognate receptor in the cytoplasm of E. coli, which makes direct comparisons between the three systems difficult. Here we show that the human CCHL (with a secretion signal) attaches heme to the human cytochrome c (with a signal sequence) in the E.coli periplasm, which is bioenergetically (p-side) analogous to the mitochondrial intermembrane space. The human CCHL is specific for the human cytochrome c, whereas recombinant system II can attach heme to multiple non-cognate c-type cytochromes (possessing the CXXCH motif.) We also show that the recombinant periplasmic systems II and III use components of the natural E.coli periplasmic DsbC/DsbD thiol-reduction pathway.

Molecular Basis Behind Inability of Mitochondrial Holocytochrome c Synthase to Mature Bacterial Cytochromes

“…In prokaryotes, many c-type cytochromes exist in addition to monoheme proteins (17)(18)(19)(20). These are all matured by Systems I and II, consistent with the proposal that only the CXXCH motif is recognized (21)(22)(23)(24). By contrast, the eukaryotic HCCS appears to require a more extended recognition domain than just the CXXCH motif for cytochrome maturation, specifically sequence features or residues located in the cyt c N terminus (see Fig.…”

“…Although the bacterial CCM system (i.e. System I) can successfully mature a wide range of mono-and multiheme cytochromes, containing simply a CX n CH motif (where X n ϭ 1-4 intervening residues) (21,24,42,43), additional structural features of eukaryotic cytochromes contribute to the strict substrate specificity of HCCS. For example, previous genetic studies have suggested that the conserved phenylalanine residue in cyt c ␣ helix-1 is critical for HCCS maturation by serving as a determinant for substrate recognition (step 2) because substitution of this residue can result in poor cyt c yields (21, 25) (Phe 11 in human cyt c; the equivalent of this position in bacterial cytochromes is not fully conserved (21, 44)).…”

Human mitochondrial holocytochrome c synthase’s heme binding, maturation determinants, and complex formation with cytochrome c

“…E. coli strains (SI Appendix, Table S2) were grown at 37°C in LB broth (Difco) supplemented with the appropriate antibiotics (Sigma-Aldrich) and other medium additives at the following concentrations, unless otherwise noted: carbenicillin, 50 μg·mL Protein Expression and Purification. For HCCS expression (with or without coexpression of cytochrome c), E. coli Δccm strain RK103 (68) or RK112 (69) was used. Starter cultures were initiated from a single colony and grown overnight at 37°C with shaking at 200 rpm in 100 mL of LB medium with the appropriate antibiotics.…”

Cytochrome c biogenesis System I: An intricate process catalyzed by a maturase supercomplex?

“…CcdA and CcsX are required for thioreduction of apocyt c thiol groups, whereas CcsAB complex transports heme b to the p side of the membrane, recognizes the apocyt c substrates and forms the thioether bonds between the apocyt c and heme b . Substrate specificity of Ccs is not yet fully established, but it may be as broad as the Ccm-System I [32, 33]. …”