A structural and functional investigation of a novel thiol-disulfide oxidoreductase

Thioredoxin (TRX) is the archetype of a family of thiol-disulfide oxidoreductases that catalyze thiol-disulfide exchange reactions in eukaryotes and prokaryotes. Thioldisulfide exchange reactions are required for a broad range of cellular processes. The thiol-disulfide status of some proteins is regulated in order to control the activity of enzymes and the function and/or stability of proteins. TRX-like oxidoreductases contain a Cys-Xaa-Xaa-Cys motif embedded in a TRX fold. The TRX fold is  ββα motif linked to βαβ motif by an α helix. Cytosolic TRX-like oxidoreductases catalyze the reduction of disulfides in a broad range of target proteins. TRX is the most abundant cytosolic thiol-disulfide oxidoreductase. TRX-like oxidoreductases are also present in oxidizing compartments of the cell ie the endoplasmic reticulum and bacterial periplasm. They catalyze either the oxidation of thiols or the reduction/isomerization of disulfides in these organelles. The ER and periplasm are sites for folding of proteins that are destined for secretion. TRX-like oxidoreductases fulfil essential roles in protein folding in these compartments by donating thiol or disulfide character to folding substrate proteins.

CcmG is a novel TRX-like oxidoreductase because unlike other TRX-like oxidoreductases that have a broad substrate specificity, CcmG has a specific oxidoreductase activity. The oxidoreductase activity of CcmG is limited to a specific reducing activity that is essential for the maturation of cytochrome c in bacteria, c- Type cytochromes are electron transfer proteins in respiratory and/or photosynthetic chains. The assembly of c-type cytochrome involves the covalent attachment of heme to reduced thiols in the Cys-Xaa-Xaa-Cys-His motif of the apocytochrome. c-Type cytochromes are assembled in the oxidizing environment of the bacterial periplasm. A specialized redox pathway has evolved in this compartment to reduce the heme binding site in the apocytochrome. This pathway involves DsbD, CcmG and CcmH. DsbD is a membrane protein that transfers electrons from TRX in the cytoplasm to periplasmic proteins, including CcmG. CcmH is a periplasmic redox protein that like CcmG, is essential for cytochrome c maturation in bacteria. Electrons are passed from TRX via the DsbD/CcmG/CcmH redox pathway to apocytochrome c so that heme attachment can occur to form holocytochrome c.

In this work, the crystal structure of B. japonicum CcmG was determined in order to understand the specialized reducing activity of CcmG. The structure of CcmG was solved by multiwavelength anomalous diffraction methods and refined to 1.14 Å resolution. The structure of CcmG includes a TRX fold that is modified to include two inserts that together form a groove near the active site. The two inserts are from discrete regions of the sequence but they come together in the structure to form a groove near the active site of CcmG. Several residues in these inserts are conserved in CcmG homologues. A comparison of the structure of CcmG with other TRX-like oxidoreductases showed that the active site of CcmG is unusually acidic. The groove and the acidic active site in the CcmG homologue in E. coli were deleted in order to investigate a possible role for these features in cytochrome c maturation. These mutant forms of ccmG were transformed into a ΔccmG E. coli strain and the levels of cytochrome c maturation were measured in an assay that detects covalently bound heme in proteins separated by SDS-PAGE. Disruption of the groove in CcmG or mutation of the acidic active site into a more hydrophobic active site disrupted cytochrome c maturation. The groove and the acidic active site in CcmG are therefore required for the function of CcmG in cytochrome c maturation. These findings demonstrate how the TRX fold, which is usually associated with a broad substrate specificity, has been adapted in CcmG to achieve a very specific function in cytochrome c maturation.

Analysis of the sequences of CcmG homologues had identified a motif ^"152 Gly-Val- Xaa-Gly-Xaa-Pro-Glu^158" as a fingerprint for CcmG homologues. The structure of CcmG showed that this motif forms a loop between the connecting α-helix and the ββα motif of the TRX fold in CcmG. This loop is very near the Cys-Xaa-Xaa-Cys active site in CcmG and has been shown in TRX to be important for protein-protein interactions. Prol57 in this motif is the cis-proline that is conserved in all TRX-like oxidoreductases identified to date and Glul58 is one of the three acidic residues that contributes to the acidic active site in CcmG.