CcsBA is a cytochrome c synthetase that also functions in heme transport.
Ontology highlight
ABSTRACT:
SUBMITTER: Frawley ER
PROVIDER: S-EPMC2700922 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Cytochrome c maturation in many bacteria, archaea, and plant mitochondria involves the integral membrane protein CcmF, which is thought to function as a cytochrome c synthetase by facilitating the final covalent attachment of heme to the apocytochrome c. We previously reported that the E. coli CcmF protein contains a b-type heme that is stably and stoichiometrically associated with the protein and is not the heme attached to apocytochrome c. Here, we show that mutation of either of two conserved transmembrane histidines (His261 or His491) impairs stoichiometric b-heme binding in CcmF and results in spectral perturbations in the remaining heme. Exogeneous imidazole is able to correct cytochrome c maturation for His261 and His491 substitutions with small side chains (Ala or Gly), suggesting that a "cavity" is formed in these CcmF mutants in which imidazole binds and acts as a functional ligand to the b-heme. The results of resonance Raman spectroscopy on wild-type CcmF are consistent with a hexacoordinate low-spin b-heme with at least one endogeneous axial His ligand. Analysis of purified recombinant CcmF proteins from diverse prokaryotes reveals that the b-heme in CcmF is widely conserved. We have also determined the reduction potential of the CcmF b-heme (E(m,7) = -147 mV). We discuss these results in the context of CcmF structure and functions as a heme reductase and cytochrome c synthetase.
| S-EPMC3237939 | biostudies-literature
Project description: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.
| S-EPMC6299221 | biostudies-literature
Project description:Strains of Escherichia coli containing mutations in the cydDC genes are defective for synthesis of the heme proteins cytochrome bd and c-type cytochromes. The cydDC genes encode a putative heterodimeric ATP-binding cassette transporter that has been proposed to act as an exporter of heme to the periplasm. To more fully understand the role of this transporter (and other factors) in heme protein biosynthesis, we developed plasmids that produce various heme proteins (e.g., cytochrome b5, cytochrome b562, and hemoglobin) in the periplasm of E. coli. By using these reporters, it was shown that the steady-state levels of polypeptides of heme proteins known to be stable without heme (e.g., cytochrome b5 and hemoglobin apoprotein) are significantly reduced in a cydC mutant. Exogenous addition of hemin to the cydC mutant still resulted in < 10% of wild-type steady-state levels of apohemoglobin in the periplasm. Since the results of heme reporter studies are not consistent with lower heme availability (i.e., heme export) in a cydC mutant, we analyzed other properties of the periplasm in cydC mutants and compared them with those of the periplasm in cydAB (encoding cytochrome bd) mutants and wild-type cells. Our results led us to favor a hypothesis whereby cydDC mutants are defective in the reduction environment within the periplasmic space. Such an imbalance could lead to defects in the synthesis of heme-liganded proteins. The heme reporters were also used to analyze strains of E. coli with a defect in genes encoding homologs of a different ABC transporter (helABC). The helABC genes have previously been shown to be required for the assembly of c-type cytochromes in Rhodobacter capsulatus (R. G. Kranz, J. Bacteriol. 171:456-464, 1989; D. L. Beckman, D. R. Trawick, and R. G. Kranz, Genes Dev. 6:268-283, 1992). This locus was shown to be essential in E. coli for endogenous cytochrome c biogenesis but not cytochrome b562 synthesis. Consistent with these and previous results, it is proposed that the HelABC transporter is specifically involved in heme export for ligation (hel). This class of periplasmic cytochromes is proposed to require heme liganding before undergoing correct folding.
| S-EPMC178509 | biostudies-other
Project description: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.
| S-EPMC8712405 | biostudies-literature
Project description:Cytochrome P450 (P450, CYP) 4A11 is a human fatty acid ω-hydroxylase that catalyzes the oxidation of arachidonic acid to the eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), which plays important roles in regulating blood pressure regulation. Variants of P450 4A11 have been associated with high blood pressure and resistance to anti-hypertensive drugs, and 20-HETE has both pro- and antihypertensive properties relating to increased vasoconstriction and natriuresis, respectively. These physiological activities are likely influenced by the redox environment, but the mechanisms are unclear. Here, we found that reducing agents (e.g. dithiothreitol and tris(2-carboxyethyl)phosphine) strongly enhanced the catalytic activity of P450 4A11, but not of 10 other human P450s tested. Conversely, added H2O2 attenuated P450 4A11 catalytic activity. Catalytic roles of five of the potentially eight implicated Cys residues of P450 4A11 were eliminated by site-directed mutagenesis. Using an isotope-coded dimedone/iododimedone-labeling strategy and mass spectrometry of peptides, we demonstrated that the heme-thiolate cysteine (Cys-457) is selectively sulfenylated in an H2O2 concentration-dependent manner. This sulfenylation could be reversed by reducing agents, including dithiothreitol and dithionite. Of note, we observed heme ligand cysteine sulfenylation of P450 4A11 ex vivo in kidneys and livers derived from CYP4A11 transgenic mice. We also detected sulfenylation of murine P450 4a12 and 4b1 heme peptides in kidneys. To our knowledge, reversible oxidation of the heme thiolate has not previously been observed in P450s and may have relevance for 20-HETE-mediated functions.
| S-EPMC5500791 | biostudies-literature
Project description:Cytochrome c(1) from mitochondrial complex III and the di-heme cytochromes c in the corresponding enzyme from epsilon-proteobacteria have so far been considered to represent unrelated cytochromes. A missing link protein discovered in the genome of the hyperthermophilic bacterium Aquifex aeolicus, however, provides evidence for a close evolutionary relationship between these two cytochromes. The mono-heme cytochrome c(1) from A. aeolicus contains stretches of strong sequence homology toward the epsilon-proteobacterial di-heme cytochromes. These di-heme cytochromes are shown to belong to the cytochrome c(4) family. Mapping cytochrome c(1) onto the di-heme sequences and structures demonstrates that cytochrome c(1) results from a mutation-induced collapse of the di-heme cytochrome structure and provides an explanation for its uncommon structural features. The appearance of cytochrome c(1) thus represents an extension of the biological protein repertoire quite different from the widespread innovation by gene duplication and subsequent diversification.
| S-EPMC539742 | biostudies-literature
Project description:Cytochrome c maturation (Ccm) is a sophisticated post-translational process. It occurs after translocation of apocytochromes c to the p side of energy transducing membranes and forms stereo-specific thioether bonds between the vinyl groups of heme b (protoporphyrin IX-Fe) and the thiol groups of cysteines at their conserved heme binding sites. In many organisms this process involves up to 10 (CcmABCDEFGHI and CcdA) membrane proteins. One of these proteins is CcmI, which has an N-terminal membrane-embedded domain with two transmembrane helices and a large C-terminal periplasmic domain with protein-protein interaction motifs. Together with CcmF and CcmH, CcmI forms a multisubunit heme ligation complex. How the CcmFHI complex recognizes its apocytochrome c substrates remained unknown. In this study, using Rhodobacter capsulatus apocytochrome c(2) as a Ccm substrate, we demonstrate for the first time that CcmI binds apocytochrome c(2) but not holocytochrome c(2). Mainly the C-terminal portions of both CcmI and apocytochrome c(2) mediate this binding. Other physical interactions via the conserved structural elements in apocytochrome c(2), like the heme ligating cysteines or heme iron axial ligands, are less crucial. Furthermore, we show that the N-terminal domain of CcmI can also weakly bind apocytochrome c(2), but this interaction requires a free thiol group at apocytochrome c(2) heme binding site. We conclude that the CcmI subunit of the CcmFHI complex functions as an apocytochrome c chaperone during the Ccm process used by proteobacteria, archaea, mitochondria of plants and red algae.
| S-EPMC3220492 | biostudies-literature
Project description:Efficient iron acquisition is critical for an invading microbe's survival and virulence. Most of the iron in mammals is incorporated into heme, which can be plundered by certain bacterial pathogens as a nutritional iron source. Utilization of exogenous heme by bacteria involves the binding of heme or hemoproteins to the cell surface receptors, followed by the transport of heme into cells. Once taken into the cytosol, heme is presented to heme oxygenases where the tetrapyrrole ring is cleaved in order to release the iron. Some Gram-negative bacteria also secrete extracellular heme-binding proteins called hemophores, which function to sequester heme from the environment. The heme-transport genes are often genetically linked as gene clusters under Fur (ferric uptake regulator) regulation. This review discusses the gene clusters and proteins involved in bacterial heme acquisition, transport and processing processes, with special focus on the heme-coordination, protein structures and mechanisms underlying heme-transport.
| S-EPMC2683585 | biostudies-literature
Project description:Heme proteins are known to perform a plethora of biologically important functions. This article reviews work that has been conducted on various class I cytochrome c proteins over a period of nearly 50 years. The article focuses on the relevance of symmetry-lowering heme–protein interactions that affect the function of the electron transfer protein cytochrome c. The article provides an overview of various, mostly spectroscopic studies that explored the electronic structure of the heme group in these proteins and how it is affected by symmetry-lowering deformations. In addition to discussing a large variety of spectroscopic studies, the article provides a theoretical framework that should enable a comprehensive understanding of the physical chemistry that underlies the function not only of cytochrome c but of all heme proteins.
| S-EPMC9781506 | biostudies-literature
Project description:A superfamily of integral membrane proteins is characterized by a conserved tryptophan-rich region (called the WWD domain) in an external loop at the inner membrane surface. The three major members of this family (CcmC, CcmF, and CcsBA) are each involved in cytochrome c biosynthesis, yet the function of the WWD domain is unknown. It has been hypothesized that the WWD domain binds heme to present it to an acceptor protein (apoCcmE for CcmC or apocytochrome c for CcmF and CcsBA) such that the heme vinyl group(s) covalently attaches to the acceptors. Alternative proposals suggest that the WWD domain interacts directly with the acceptor protein (e.g., apoCcmE for CcmC). Here, it is shown that CcmC is only trapped with heme when its cognate acceptor protein CcmE is present. It is demonstrated that CcmE only interacts stably with CcmC when heme is present; thus, specific residues in each protein provide sites of interaction with heme to form this very stable complex. For the first time, evidence that the external WWD domain of CcmC interacts directly with heme is presented. Single and multiple substitutions of completely conserved residues in the WWD domain of CcmC alter the spectral properties of heme in the stable CcmC:heme:CcmE complexes. Moreover, some mutations reduce the binding of heme up to 100%. It is likely that endogenously synthesized heme enters the external WWD domain of CcmC either via a channel within this six-transmembrane-spanning protein or from the membrane. The data suggest that a specific heme channel (i.e., heme binding site within membrane spanning helices) is not present in CcmC, in contrast to the CcsBA protein. We discuss the likelihood that it is not important to protect the heme via trafficking in CcmC whereas it is critical in CcsBA.
| S-EPMC3580838 | biostudies-literature