Cytochrome bc1 Complex (PDB ID:1BCC from Gallus gallus
Created by: Christi Otero
Cytochrome bc1 complex (PBD 1BCC), also commonly known as ubiquinol-cytochrome c reductase of Complex III, from Gallus gallus is classified as an oxidoreductase and is one of three major respiratory enzymes complexes residing in the inner mitochondrial membrane (12).
Involved in the electron transport chain, Cytochrome bc1 complex is the most conserved electron transport complex capable of energy transduction (11). The complex is found in the plasma membrane of phylogenetically diverse and respiring bacteria and is located in the inner mitochondrial membrane of all eukaryotic cells. In all species, Cytochrome bc1 complex transfers electrons from low-potential ubiquinol, a carrier of hydrogen atoms found in the mitochondrial membrane, to a higher potential cytochrome c via a unique redox pathway called the Q-cycle (5). The Q-cycle, couples electron transfer to proton translocation and generates a proton gradient. Cytochrome bc1 complex plays an essential role in the biochemical generation of ATP via oxidative phosphorylation (11).
The complex uses three different electron transfer cytochrome proteins which contain four redox prosthetic groups. The components of cytochrome bc1 are; a b-type cytochrome which contains two noncovalently bound heme groups (11); cytochrome c1, which contains a covalently bound c-type heme group attached to the protein through thiol ether linkages (11); and the Rieske iron-sulfur protein, which contains a 2Fe-2S cluster coordinately liganded to two cysteine and two histidine (11). Analysis of the crystal structure of cytochrome bc1 complex reveals that the ISP may undergo significant movement during the catalytic cycle (3)
Crystals from multiple sources have confirmed cytochrome bc1 complex is a dimeric protein in which two monomers are related by a two-fold axis perpendicular to the membrane (12). The protein extends 79Å from the membrane into the matrix space and 31Å into the intermembrane region on either side of the transmembrane region, 40Å thick. Together, the length of cytochrome bc1 complex is 150Å perpendicular to the membrane. (12) The complex from chicken is comprised of 2015 residues,10 subunits, with a molecular weight of 242742 Da and a theoretical isoelectric point of 6.51(6). The 10 subunits of cytochrome bc1 complex of chicken do not share the same secondary structure. Subunit 1 is 45% α-helices and 18% ß sheets. Subunit 2 is 41% α helices and 18% ß sheets. Subunit 3, cytochrome b, is 65% α helices and 2% ß sheets. Subunit 4, cytochrome c1, is 39% α helices and 7% ß sheets. Subunit 5, Rieske Iron-Sulfur Protein, is 29% α helices and 22% ß sheets. Subunit 6 is 61% α helices. Subunit 7, Q-binding site, is 41% α helices and 9% ß sheets. Subunit 8, c1 "hinge", is 61% α helices. Subunit 9, Fe-S presequence, has not been sequenced. And lastly, Subunit 10 is 74% α helices. Taken together, the cytochrome bc1 complex from chicken is comprised of 45.6% α helices and 12.7% ß sheets.
Four prosthetic groups per monomer of cytochrome bc1 have been identified. The prosthetic groups include one 2Fe-2S cluster and three hemes (heme bL and heme bH of cyt b andheme c of cyt c1). A heme is a prosthetic group containing an iron atom in the center of a heterocyclic ring. The two hemes on the b cytochrome are structurally conserved among various species. The two heme groups are distinguished by their reduction potentials and wavelengths. Heme bL of cytochrome b has a standard reduction potential of -0.100 V and maximal absorbance of 566 nm. Heme bH of cytochrome b has a standard reduction potential of +0.050V and a maximal absorbance of 562 nm (5).
The ISP and cytochrome c1 are similar in structure; each contains a globular domain and is anchored to the inner mitochondrial membrane by a hydrophobic segment. The two structures differ in that the hydrophobic segment of the Rieske (ISP) protein is on the N-terminal and C-terminal in cytochrome c1 (5).
As mentioned, the iron-sulfur protein has a hydrophobic membrane-spanning helical segment near the N-terminus. The transmembrane helix of the Rieske protein is slightly curved and highly slanted, passing through the membrane at a 32° angle perpendicular to the membrane (12). At the tip of the protein, two histidine ligands, residues 141 and 161, are viewed as bulges (12). Residue H161of the Rieske protein provides one of the ligands to the 2Fe-2S cluster.
Comparison between the structure of cytochrome bc1 complex from chicken in the presence and absence of stigmatellin inhibitors has provided insight that the extrinsic domain of the functionally important Rieske iron-sulfur protein assumes one of two conformations. In one conformation, the iron-sulfur cluster is near the heme group of cytochrome c1, but far from the binding site of ubiquinol in cytochrome b. In the second conformation, the iron-sulfur cluster is closer to cytochrome b and further from cytochrome c1 (12). The two locations are related by a 57° rotation (12). It has been proposed that the reaction mechanism for electron transfer in cytochrome bc1 complex requires a dramatic conformational change involving movement of the iron-sulfur protein due to the two conformations observed (12).
Cytochrome c1 is another functionally important redox-active protein of the cytochrome bc1 complex. The extrinsic domain of cytochrome c1 forms a wedge-like structure containing the c-type heme group, with a C-terminal transmembrane anchor next to cytochrome b (12). Three α helices (1, 3 and 5) from class I cytochrome in general, are present in cytochrome c1 and occupy the same positions. Conserved aromatic residues involved in the interaction between α 1 and α 5 are present as Y33 and F189, respectively (12). Furthermore, the tripeptide PNL is highly conserved in almost all cytochromes c1. The proline carbonyl accepts a hydrogen bond from the histidine heme ligand and leucine provides a hydrophobic environment for the heme ring.
PEWY motif is found in hundreds of cytochrome b sequences. The glutamic acid in the PEWY loop is involved in inhibitor binding at the Qo site and functions to moderate rate of ubiquinol (UQH2) oxidation (as shown by the chicken cyt bc1 cocrystallized with stigmatellin pdb-2bcc). It has been reported that mutants of the PEWY loop have a decreased rate of oxidation in those with glutamate replaced by other residues (10). Given this finding, it has been inferred that hydrogen bonding of ubiquinol to glutamate stabilizes the binding of quinol to the site
The cytochrome bc1 complex functions through a Q-cycle mechanism which is accompanied by proton transport across the inner mitochondrial membrane. The suggested pathway for electrons in this system is shown here. (5) Cytochrome bc1 complex catalyzes the reduction of cytochrome c by oxidation of coenzyme Q (CoQ) while simultaneously pumping four protons from the mitochondrial matrix to the intermembrane space. During the Q cycle, two protons are used from the matrix or negative (N) side; four protons (H+) are released into the intermembrane or positive (P) side; and two electrons are passed to cytochrome c. As a result of the Q-cycle, a proton gradient is formed across the membrane (5).
Both the oxidation and reduction of ubiquinol and ubiquinone, respectively, occur in the cytochrome bc1 complex at physically distinct locations. A pool of ubiquinone (UQ) and ubiquinol (UQH2) exists in the inner mitochondrial membrane. The Q-cycle is initiated when a molecule of UQH2 from the pool enters into the Qp-site on the bc1 complex near the cystolic face of the membrane (5). Oxidation of the UQH2 occurs in two steps. First, an electron from UQH2 is transferred to the ISP and secondly to cytochrome c1 (5). It is hypothesized that the extrinsic domain of the ISP is mobile and shifts between two different catalytic sites. The mobility of ISP enables it to oxidize the quinol bound in the Qp-site within the cytochrome b and reduce the heme bound in the cytochrome c1.(4) This releases two protons to the cytosol and leaves UQ.-, a semiquinone anion form of UQ, at the Qp-site. The semiquinone anion produced is oxidized at the Qp-site via the transfer of the second electron to the bL heme of cytochrome b, converting the semiquinone anion to ubiquinone (5). The electron on the bL heme (facing the cytosol) is passed to the bH heme on the matrix side of the membrane. The transfer of the electron occurs against a membrane potential of 0.15V and is driven by the loss of redox potential as the electron passes from bL to bH. The electron then passes from bH to a molecule of UQ at a second quinone-binding site, Qn, which converts the ubiquinone to semiquinone anion. The anion is strongly bound to the Qn-site, thus completing the first half of the Q cycle (5).
The second half of the Q cycle is similar to the first half. The second molecule of ubiquinol is oxidized at the Qp site such that one electron is passed to cytochrome c1 and the other is transferred to heme bL and then heme bH. The bH electron is transferred to the semiquinone anion at the Qn site (5). The coupling to proton transfer depends on a bifurcated reaction at the Qp-site of the complex, in which the two electrons from ubiquinol are passed to cytochrome c1. The cycle is completed when two protons from the mitochondrial matrix produce a molecule of ubiquinol which is released from the Qn site to return to the coenzyme Q pool (5).
The overall shape of the cytochrome bc1 complex dimer of chicken is similar to the complex described from bovine. Each monomer of bovine consists of 2165 amino acid residues and 11 protein subunits (5). Subunit 11, however, does not seem to be present in the chicken enzyme. Subunit 9 has yet to be characterized. This subunit is the presequence of the Rieske protein and is lodged in between the two core subunits at the matrix side of the complex (8), These core subunits are cleaved off by matrix-located processing protease (2) and the structure reveals the mechanism by which mitochondrial targeting presequences are recognized (8). Subunit 8 has been characterized as the ‘hinge protein for the formation of the cytochrome c1-c complex (9) and the external ends of subunits 7 and 10 interact with cytochrome c1, opposite from the dimer interface. The hinge protein consists of a bent hairpin held by two internal disulfide bonds (12). Taken together, the 10 subunits characterized in the chicken correspond to 3 respiratory subunits (cytochrome b, cytochrome c1 and Rieske protein), 2 core proteins and 5 low molecular weight proteins.
Subunit 1, Cytochrome bc1 complex from Bos taurus (1BGY-A) is 90% similar to Subunit 1 from Gallus gallus. Cytochrome bc1 complex from bovine contains 446 amino acid residues and the complex from chicken is composed of 442 amino acid residues. The two complexes differ in secondary structure. Subunit 1 of cytochrome bc1 from chicken is composed of 45% α helices (23 helices, 201 residues) and 18% ß sheets (15 strands, 82 residues). By comparison, subunit 1 of cytochrome bc1 from bovine is composed of 45% α helices (23 helices, 205 residues) and 15% ß sheets (14 strands, 71 residues). Furthermore, as mentioned above, the cytochrome bc1 from chicken has 10 subunits - subunit 11 seems not to be present in the chicken enzyme. The function of the additional subunits are generally not well known and are being actively explored by genetically manipulating proteins in various species (11)
The use of various bioinformatics tools aid in the identification of protein structure. The Dali server uses a sum-of-pairs method, which measures similarity by comparing intramolecular distances (6). The similarity between structures is measured by Dali-Z scores in which a Z-score greater than two signifies proteins with similar tertiary structure. The Dali server thus, provided a comparison protein based on a protein query with similar tertiary structure and the results of DALI (Z score=51.6) show that Cytochrome bc1 complex from chicken (1BCC) has tertiary structure similarity to the cytochrome bc1 complex from bovine (1BGY-A) (7). PSI-BLAST runs a protein query for proteins of similar primary structure. An E-value is determined based on gaps of amino acids that the query does not share with the subject protein. The E-value decreases with increase sequence homology and increases with the presence of gaps. An E-value less than 0.05 indicates significant similarity in primary structure. PSI-BLAST comparison between bc1 complex from chicken and bc1 complex from bovine produced an E-value of 0.0. The small E-value signifying that the primary structure of cytochrome bc1 from bovine is homologous to the subject protein, cytochrome bc1 from chicken (1). ExPASy is a bioinformatics resource that provides access to scientific resources databases and software tools in different areas of life sciences. One of the tools provided by ExPASy is the computation of theoretical isoelectric point and molecular weight, both of which were indicated above (6).
The cytochrome bc1 complex from chicken is a good model for the cytochrome bc1 complex residing in the inner mitochondrial membrane. Across phylogenetically diverse species, cytochrome bc1 complex has been highly conserved and various species depict similar architecture - especially in the three electron transporting cytochromes and Rieske iron-sulfur cluster Knockout studies depict differences in binding abilities and rate of electron transport. Understanding and mapping the structure of cytochrome bc1 complex of chicken provides a refined model of the oxidoreductase involved in the electron transport chain. Through the crystallization of the complex from chicken, a better understanding of the structure and function of various subunits has become available.