Andreia F. Verissimo

CcmI Subunit of CcmFHI Heme Ligation Complex Functions as an Apocytochrome c Chaperone during c-Type Cytochrome Maturation

Background: Cytochrome c maturation (Ccm) is the covalent ligation of heme b to an apocytochrome c by the Ccm apparatus. Results: CcmI subunit of CcmFHI heme ligation complex recognizes and binds specifically apo- and not holocytochrome c2. Conclusion: CcmI and its homologues are apocytochrome c chaperones. Significance: A first glimpse to how the heme ligation complex recognizes an apocytochrome c before, and releases a holocytochrome c after, cofactor addition. 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 c2 as a Ccm substrate, we demonstrate for the first time that CcmI binds apocytochrome c2 but not holocytochrome c2. Mainly the C-terminal portions of both CcmI and apocytochrome c2 mediate this binding. Other physical interactions via the conserved structural elements in apocytochrome c2, 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 c2, but this interaction requires a free thiol group at apocytochrome c2 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.

The Heme Chaperone ApoCcmE Forms a Ternary Complex with CcmI and Apocytochrome c

Background: Cytochrome c maturation (Ccm) is the covalent ligation of heme b to an apocytochrome c. Results: CcmE forms a stable ternary complex with CcmI and apocytochrome c. Conclusion: Together with CcmFHI, the heme chaperone CcmE is a part of the heme ligation complex that matures apocytochromes c. Significance: These findings contribute to our mechanistic understanding of how the Ccm process occurs in cells. Cytochrome c maturation (Ccm) is a post-translational process that occurs after translocation of apocytochromes c to the positive (p) side of energy-transducing membranes. Ccm is responsible for the formation of covalent bonds between the thiol groups of two cysteines residues at the heme-binding sites of the apocytochromes and the vinyl groups of heme b (protoporphyrin IX-Fe). Among the proteins (CcmABCDEFGHI and CcdA) required for this process, CcmABCD are involved in loading heme b to apoCcmE. The holoCcmE thus formed provides heme b to the apocytochromes. Catalysis of the thioether bonds between the apocytochromes c and heme b is mediated by the heme ligation core complex, which in Rhodobacter capsulatus contains at least the CcmF, CcmH, and CcmI components. In this work we show that the heme chaperone apoCcmE binds to the apocytochrome c and the apocytochrome c chaperone CcmI to yield stable binary and ternary complexes in the absence of heme in vitro. We found that during these protein-protein interactions, apoCcmE favors the presence of a disulfide bond at the apocytochrome c heme-binding site. We also establish using detergent-dispersed membranes that apoCcmE interacts directly with CcmI and CcmH of the heme ligation core complex CcmFHI. Implications of these findings are discussed with respect to heme transfer from CcmE to the apocytochromes c during heme ligation assisted by the core complex CcmFHI.

Uncovering the Transmembrane Metal Binding Site of the Novel Bacterial Major Facilitator Superfamily-Type Copper Importer CcoA

ABSTRACT Uptake and trafficking of metals and their delivery to their respective metalloproteins are important processes. Cells need precise control of each step to avoid exposure to excessive metal concentrations and their harmful consequences. Copper (Cu) is a required micronutrient used as a cofactor in proteins. However, in large amounts, it can induce oxidative damage; hence, Cu homeostasis is indispensable for cell survival. Biogenesis of respiratory heme-Cu oxygen (HCO) reductases includes insertion of Cu into their catalytic subunits to form heme-Cu binuclear centers. Previously, we had shown that CcoA is a major facilitator superfamily (MFS)-type bacterial Cu importer required for biogenesis of cbb3-type cytochrome c oxidase (cbb3-Cox). Here, using Rhodobacter capsulatus, we focused on the import and delivery of Cu to cbb3-Cox. By comparing the CcoA amino acid sequence with its homologues from other bacterial species, we located several well-conserved Met, His, and Tyr residues that might be important for Cu transport. We determined the topology of the transmembrane helices that carry these residues to establish that they are membrane embedded, and substituted for them amino acids that do not ligand metal atoms. Characterization of these mutants for their uptake of radioactive 64Cu and cbb3-Cox activities demonstrated that Met233 and His261 of CcoA are essential and Met237 and Met265 are important, whereas Tyr230 has no role for Cu uptake or cbb3-Cox biogenesis. These findings show for the first time that CcoA-mediated Cu import relies on conserved Met and His residues that could act as metal ligands at the membrane-embedded Cu binding domain of this transporter. IMPORTANCE Cu is a micronutrient that is both essential and toxic; hence, its cellular homeostasis is crucial. Respiratory cbb3-type cytochrome c oxidases (cbb3-Cox) are Cu-containing energy-transducing enzymes that are important for many microaerophilic processes, including photosynthesis, respiration, and bacterial pathogenesis. How Cu is incorporated into cbb3-Cox enzymes is not well known. So far, CcoA is the only known major facilitator superfamily (MFS)-type transporter required for Cu import into the bacterial cytoplasm and for cbb3-Cox biogenesis. This study shows that the membrane-embedded, universally conserved Met and His residues of CcoA are essential for its Cu import function and also for its role in cbb3-Cox biogenesis, shedding light on the mechanism of function of this bacterial prototypical Cu importer. Cu is a micronutrient that is both essential and toxic; hence, its cellular homeostasis is crucial. Respiratory cbb3-type cytochrome c oxidases (cbb3-Cox) are Cu-containing energy-transducing enzymes that are important for many microaerophilic processes, including photosynthesis, respiration, and bacterial pathogenesis. How Cu is incorporated into cbb3-Cox enzymes is not well known. So far, CcoA is the only known major facilitator superfamily (MFS)-type transporter required for Cu import into the bacterial cytoplasm and for cbb3-Cox biogenesis. This study shows that the membrane-embedded, universally conserved Met and His residues of CcoA are essential for its Cu import function and also for its role in cbb3-Cox biogenesis, shedding light on the mechanism of function of this bacterial prototypical Cu importer.

Engineering a prokaryotic apocytochrome c as an efficient substrate for Saccharomyces cerevisiae cytochrome c heme lyase

Cytochromes c are heme proteins that require multiple maturation components, such as heme lyases, for cofactor incorporation. Saccharomyces cerevisiae has two heme lyases that are specific for apocytochromes c (CCHL) or c(1) (CC(1)HL). CCHL can covalently attach heme b groups to apocytochrome c substrates of eukaryotic but not prokaryotic origin. Besides their conserved Cys-Xxx-Xxx-Cys-His heme-binding motifs, the amino-terminal regions of apocytochrome c substrates appear to be important for CCHL function. In this study, we show for the first time that only two amino acid changes in the amino-terminal region of the non-CCHL substrate apocytochrome c(2) from Rhodobacter capsulatus are necessary and sufficient for efficient holocytochrome c formation by CCHL. This finding led us to propose a consensus sequence located at the amino-terminus of apocytochromes c, and critical for substrate recognition and heme ligation by CCHL.

Widespread Distribution and Functional Specificity of the Copper Importer CcoA: Distinct Cu Uptake Routes for Bacterial Cytochrome c Oxidases

ABSTRACT Cytochrome c oxidases are members of the heme-copper oxidase superfamily. These enzymes have different subunits, cofactors, and primary electron acceptors, yet they all contain identical heme-copper (CuB) binuclear centers within their catalytic subunits. The uptake and delivery pathways of the CuB atom incorporated into this active site, where oxygen is reduced to water, are not well understood. Our previous work with the facultative phototrophic bacterium Rhodobacter capsulatus indicated that the copper atom needed for the CuB site of cbb3-type cytochrome c oxidase (cbb3-Cox) is imported to the cytoplasm by a major facilitator superfamily-type transporter, CcoA. In this study, a comparative genomic analysis of CcoA orthologs in alphaproteobacterial genomes showed that CcoA is widespread among organisms and frequently co-occurs with cytochrome c oxidases. To define the specificity of CcoA activity, we investigated its function in Rhodobacter sphaeroides, a close relative of R. capsulatus that contains both cbb3- and aa3-Cox. Phenotypic, genetic, and biochemical characterization of mutants lacking CcoA showed that in its absence, or even in the presence of its bypass suppressors, only the production of cbb3-Cox and not that of aa3-Cox was affected. We therefore concluded that CcoA is dedicated solely to cbb3-Cox biogenesis, establishing that distinct copper uptake systems provide the CuB atoms to the catalytic sites of these two similar cytochrome c oxidases. These findings illustrate the large variety of strategies that organisms employ to ensure homeostasis and fine control of copper trafficking and delivery to the target cuproproteins under different physiological conditions. IMPORTANCE The cbb3- and aa3-type cytochrome c oxidases belong to the widespread heme-copper oxidase superfamily. They are membrane-integral cuproproteins that catalyze oxygen reduction to water under hypoxic and normoxic growth conditions. These enzymes diverge in terms of subunit and cofactor composition, yet they all share a conserved heme-copper binuclear site within their catalytic subunit. In this study, we show that the copper atoms of the catalytic center of two similar cytochrome c oxidases from this superfamily are provided by different copper uptake systems during their biogenesis. This finding illustrates different strategies by which organisms fine-tune the trafficking of copper, which is an essential but toxic micronutrient. The cbb3- and aa3-type cytochrome c oxidases belong to the widespread heme-copper oxidase superfamily. They are membrane-integral cuproproteins that catalyze oxygen reduction to water under hypoxic and normoxic growth conditions. These enzymes diverge in terms of subunit and cofactor composition, yet they all share a conserved heme-copper binuclear site within their catalytic subunit. In this study, we show that the copper atoms of the catalytic center of two similar cytochrome c oxidases from this superfamily are provided by different copper uptake systems during their biogenesis. This finding illustrates different strategies by which organisms fine-tune the trafficking of copper, which is an essential but toxic micronutrient.

Biogenesis of Cytochrome c Complexes: From Insertion of Redox Cofactors to Assembly of Different Subunits

Cytochromes (cyts) are ubiquitous heme containing proteins that are key components of energy transduction pathways. They participate in a wide variety of electron transfer reactions, which are essential for cellular processes responsible for chemical energy (ATP) production. The cbb3-type cyt c oxidase (cbb3-Cox) provides an excellent model to study biogenesis of membrane-integral, oligomeric cyt c complexes. Its subunits contain three hemes c, two hemes b and a copper (CuB) atom as cofactors that use distinct insertion processes. In cyts c, heme b is covalently ligated (referred to as heme c) via a complex maturation process that involves in some species up to nine components (Ccm-System I). In addition to the cyts c, many cyt c containing complexes carry other cofactors, and insertion of these cofactors requires additional biogenesis components besides the Ccm-system I. In the case of cbb3-Cox, the mechanisms underlying incorporation of hemes b into the catalytic subunit are not well understood. However, remarkable progress was achieved recently on how the single CuB atom at the catalytic heart of this heme-copper oxidase is acquired. Finally, insertion of the cofactors must be temporally and spatially coordinated with the assembly of the subunits in order to yield a functional cbb3-Cox enzyme. In this chapter, we discuss the biogenesis of cbb3-Cox from the insertion of its catalytic heme-copper (CuB) center and maturation of its c-type cyts to the assembly of its mature subunits, mainly focusing on studies carried out with the anoxygenic phototrophic bacterium Rhodobacter capsulatus.

During Cytochrome c Maturation CcmI Chaperones the Class I Apocytochromes until the Formation of Their b-Type Cytochrome Intermediates

Background: Cytochrome c maturation (Ccm) forms thioether bonds between heme b and c-type apocytochromes. Results: CcmI chaperone exhibits a very high binding affinity for the class I c-type apocytochromes, which decreases drastically in the presence of heme. Conclusion: CcmI holds the c-type apocytochromes tightly until heme coordination yields their b-type intermediates during Ccm. Significance: Interactions between the c-type apocytochromes and chaperones are critical for the Ccm process. The c-type cytochromes are electron transfer proteins involved in energy transduction. They have heme-binding (CXXCH) sites that covalently ligate heme b via thioether bonds and are classified into different classes based on their protein folds and the locations and properties of their cofactors. Rhodobacter capsulatus produces various c-type cytochromes using the cytochrome c maturation (Ccm) System I, formed from the CcmABCDEFGHI proteins. CcmI, a component of the heme ligation complex CcmFHI, interacts with the heme-handling protein CcmE and chaperones apocytochrome c2 by binding its C-terminal helix. Whether CcmI also chaperones other c-type apocytochromes, and the effects of heme on these interactions were unknown previously. Here, we purified different classes of soluble and membrane-bound c-type apocytochromes (class I, c2 and c1, and class II c′) and investigated their interactions with CcmI and apoCcmE. We report that, in the absence of heme, CcmI and apoCcmE recognized different classes of c-type apocytochromes with different affinities (nm to μm KD values). When present, heme induced conformational changes in class I apocytochromes (e.g. c2) and decreased significantly their high affinity for CcmI. Knowing that CcmI does not interact with mature cytochrome c2 and that heme converts apocytochrome c2 into its b-type derivative, these findings indicate that CcmI holds the class I apocytochromes (e.g. c2) tightly until their noncovalent heme-containing b-type cytochrome-like intermediates are formed. We propose that these intermediates are subsequently converted into mature cytochromes following the covalent ligation of heme via the remaining components of the Ccm complex.

Absence of Thiol-Disulfide Oxidoreductase DsbA Impairs cbb3-Type Cytochrome c Oxidase Biogenesis in Rhodobacter capsulatus

The thiol-disulfide oxidoreductase DsbA carries out oxidative folding of extra-cytoplasmic proteins by catalyzing the formation of intramolecular disulfide bonds. It has an important role in various cellular functions, including cell division. The purple non-sulfur bacterium Rhodobacter capsulatus mutants lacking DsbA show severe temperature-sensitive and medium-dependent respiratory growth defects. In the presence of oxygen, at normal growth temperature (35°C), DsbA− mutants form colonies on minimal medium, but they do not grow on enriched medium where cells elongate and lyse. At lower temperatures (i.e., 25°C), cells lacking DsbA grow normally in both minimum and enriched media, however, they do not produce the cbb3-type cytochrome c oxidase (cbb3-Cox) on enriched medium. Availability of chemical oxidants (e.g., Cu2+ or a mixture of cysteine and cystine) in the medium becomes critical for growth and cbb3-Cox production in the absence of DsbA. Indeed, addition of Cu2+ to the enriched medium suppresses, and conversely, omission of Cu2+ from the minimal medium induces, growth and cbb3-Cox defects. Alleviation of these defects by addition of redox-active chemicals indicates that absence of DsbA perturbs cellular redox homeostasis required for the production of an active cbb3-Cox, especially in enriched medium where bioavailable Cu2+ is scarce. This is the first report describing that DsbA activity is required for full respiratory capability of R. capsulatus, and in particular, for proper biogenesis of its cbb3-Cox. We propose that absence of DsbA, besides impairing the maturation of the c-type cytochrome subunits, also affects the incorporation of Cu into the catalytic subunit of cbb3-Cox. Defective high affinity Cu acquisition pathway, which includes the MFS-type Cu importer CcoA, and lower production of the c-type cytochrome subunits lead together to improper assembly and degradation of cbb3-Cox.