Cryo-EM of CcsBA reveals the basis for cytochrome c biogenesis and heme transport.

Although the individual structures and respiratory functions of cytochromes are well studied, the structural basis for their assembly, including transport of heme for attachment, are unknown. We describe cryo-electron microscopy (cryo-EM) structures of CcsBA, a bifunctional heme transporter and cytochrome c (cyt c) synthase. Models built from the cryo-EM densities show that CcsBA is trapped with heme in two conformations, herein termed the closed and open states. The closed state has heme located solely at a transmembrane (TM) site, with a large periplasmic domain oriented such that access of heme to the cytochrome acceptor is denied. The open conformation contains two heme moieties, one in the TM-heme site and another in an external site (P-heme site). The presence of heme in the periplasmic site at the base of a chamber induces a large conformational shift that exposes the heme for reaction with apocytochrome c (apocyt c). Consistent with these structures, in vivo and in vitro cyt c synthase studies suggest a mechanism for transfer of the periplasmic heme to cytochrome.

In vitro reconstitution reveals major differences between human and bacterial cytochrome c synthases.

Cytochromes c are ubiquitous heme proteins in mitochondria and bacteria, all possessing a CXXCH (CysXxxXxxCysHis) motif with covalently attached heme. We describe the first in vitro reconstitution of cytochrome c biogenesis using purified mitochondrial (HCCS) and bacterial (CcsBA) cytochrome c synthases. We employ apocytochrome c and peptide analogs containing CXXCH as substrates, examining recognition determinants, thioether attachment, and subsequent release and folding of cytochrome c. Peptide analogs reveal very different recognition requirements between HCCS and CcsBA. For HCCS, a minimal 16-mer peptide is required, comprised of CXXCH and adjacent alpha helix 1, yet neither thiol is critical for recognition. For bacterial CcsBA, both thiols and histidine are required, but not alpha helix 1. Heme attached peptide analogs are not released from the HCCS active site; thus, folding is important in the release mechanism. Peptide analogs behave as inhibitors of cytochrome c biogenesis, paving the way for targeted control.

From tiny bacteria to the tallest trees, most life on Earth carries a protein called cytochrome c, which helps to create the energy that powers up cells. Cytochrome c does so thanks to its heme, a molecule that enables the chemical reactions required for the energy-creating process. Despite both relying on cytochrome c, animals and bacteria differ in the enzyme they use to attach the heme to the cytochrome. Spotting variations in how this 'cytochrome c synthase' works would help to find compounds that deactivate the enzyme in bacteria, but not in humans. However, studying cytochrome c synthase in living cells is challenging. To bypass this issue, Sutherland, Mendez, Babbitt et al. successfully reconstituted cytochrome c synthases from humans and bacteria in test tubes. This allowed them to examine in detail which structures the enzymes recognize to spot where to attach the heme onto their target. The experiments revealed that human and bacterial synthases actually rely on different parts of the cytochrome c to orient themselves. Different short compounds could also block either the human or bacterial enzyme. Variations between human and bacterial cytochrome c synthase could lead to new antibiotics which deactivate the cytochrome and kill bacteria while sparing patients. The next step is to identify molecules that specifically interfere with cytochrome c synthase in bacteria, and could be tested in clinical trials.

Structure-Function Analysis of the Bifunctional CcsBA Heme Exporter and Cytochrome c Synthetase.

Although intracellular heme trafficking must occur for heme protein assembly, only a few heme transporters have been unequivocally discovered and nothing is known about their structure or mechanisms. Cytochrome c biogenesis in prokaryotes requires the transport of heme from inside to outside for stereospecific attachment to cytochrome c via two thioether bonds (at CXXCH). The CcsBA integral membrane protein was shown to transport and attach heme (and thus is a cytochrome c synthetase), but the structure and mechanisms underlying these two activities are poorly understood. We employed a new cysteine/heme crosslinking tool that traps endogenous heme in heme binding sites. We combined these data with a comprehensive imidazole correction approach (for heme ligand interrogation) to map heme binding sites. Results illuminate the process of heme transfer through the membrane to an external binding site (called the WWD domain). Using meta-genomic data (GREMLIN) and Rosetta modeling programs, a structural model of the transmembrane (TM) regions in CcsBA were determined. The heme mapping data were then incorporated to model the TM heme binding site (with TM-His1 and TM-His2 as ligands) and the external heme binding WWD domain (with P-His1 and P-His2 as ligands). Other periplasmic structure/function studies facilitated modeling of the full CcsBA protein as a framework for understanding the mechanisms. Mechanisms are proposed for heme transport from TM-His to WWD/P-His and subsequent stereospecific attachment of heme. A ligand exchange of the P-His1 for histidine of CXXCH at the synthetase active site is suggested.IMPORTANCE The movement or trafficking of heme is critical for cellular functions (e.g., oxygen transport and energy production); however, intracellular heme is tightly regulated due to its inherent cytotoxicity. These factors, combined with the transient nature of transport, have resulted in a lack of direct knowledge on the mechanisms of heme binding and trafficking. Here, we used the cytochrome c biogenesis system II pathway as a model to study heme trafficking. System II is composed of two integral membrane proteins (CcsBA) which function to transport heme across the membrane and stereospecifically position it for covalent attachment to apocytochrome c We mapped two heme binding domains in CcsBA and suggest a path for heme trafficking. These data, in combination with metagenomic coevolution data, are used to determine a structural model of CcsBA, leading to increased understanding of the mechanisms for heme transport and the cytochrome c synthetase function of CcsBA.