Subunit IV of Cytochrome bc 1 Complex from Rhodobacter sphaeroides

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To confirm that the cytochrome bc 1 complex exists as a dimer with intertwining Rieske iron-sulfur proteins in solution, four Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc 1 complexes containing two pairs of cysteine substitutions, one in the interface between the head domain of iron-sulfur protein (ISP) and cytochrome b and the other between the tail domain of ISP and cytochrome b, were generated and characterized. The cytochrome bc 1 complex (also known as ubiquinol-cytochrome c reductase or Complex III) is an essential segment of the electron transfer chains of mitochondria and many respiratory and photosynthetic bacteria (1). This complex catalyzes electron transfer from ubiquinol to cytochrome c and concomitantly translocates protons across the membrane to generate a membrane potential and pH gradient for ATP synthesis. The polypeptide composition of cytochrome bc 1 complexes from different sources varies from three to eleven subunits. The redox subunits: cytochrome b, cytochrome c 1 , and the Rieske ironsulfur protein (ISP 1 ), are conserved in all cytochrome bc 1 complexes.Recently, mitochondrial cytochrome bc 1 complexes from beef (2, 3), chicken (4), and yeast (5) were crystallized and their three-dimensional structure determined. These three-dimensional structures not only established the location of the redox centers, transmembrane helices, and inhibitor binding sites, but also showed an unexpected dimeric structural arrangement of this complex in the crystal (2-5). The two ISPs from the two monomers are intertwined; the head domain of ISP in one monomer is close to the cytochrome b and cytochrome c 1 in the other monomer. The molecule of ISP can be divided into three domains: head, tail, and neck, with the 2Fe-2S cluster located at the tip of the head (6, 7). In tetragonal I4 1 22 crystals of native oxidized bovine cytochrome bc 1 complex, the 2Fe-2S cluster in one monomer is 27 Å from heme b L of the other monomer and 40 Å from the heme b L of the same monomer (2). This structural arrangement suggests that mitochondrial bc 1 complex functions as a dimer, because the distance between the 2Fe-2S of ISP of one monomer and the low potential cytochrome b (b L ) of the other monomer is less than that between these groups in the same monomer. The shorter distance accommodates fast electron transfer from QH 2 to ISP and b L . However, a complex that exists as a dimer in the crystal might exist and function as a monomer in solution. Evidence for the cytochrome bc 1 complex functioning as a dimer or a monomer has been reported (8 -11). Therefore, it is important to establish whether or not the structure of the dimeric cytochrome bc 1 complex observed in the crystal also exists in solution.One way to address this question is to generate a mutant bc 1 complex that forms two intersubunit disulfide bonds, one at the interface between the head domain of ISP and cytochrome b and the other at the proximity of the tail domain of ISP and cytochrome b. If the complex exists as a dimer with intertw...

Section: Methodsmentioning

confidence: 99%

Evidence for the Intertwined Dimer of the Cytochrome bc 1 Complex in Solution

Xiao

Chandrasekaran²,

Yu³

2001

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“…2, lanes This lack of intersubunit disulfide bond formation between an engineered cysteine in cytochrome b or ISP and an endogenous cysteine in ISP or cytochrome b is as expected. The R. sphaeroides cytochrome bc 1 complex has nine endogenous cysteine residues: one in cytochrome b, four in cytochrome c 1 (21). They form an intrasubunit disulfide bond that brings the two loops together and stabilizes this region.…”

Section: Photosynthetic Growth Behaviors Of Mutants Carrying Cysteinementioning

confidence: 99%

“…These choices were based on the three-dimensional structural model of the four-subunit cytochrome bc 1 complex of R. sphaeroides (Fig. 1A) constructed with coordinates from bovine cytochromes b and c 1 , ISP, and subunit XII (21). The distances between these three cysteine pairs are 6.1, 6.1, and, 7.5 Å, respectively, in the bacterial complex (Fig.…”

Section: Determination Of Electron Transfer Rates Between the 2fe2s Cmentioning

confidence: 99%

Confirmation of the Involvement of Protein Domain Movement during the Catalytic Cycle of the Cytochrome bc1Complex by the Formation of an Intersubunit Disulfide Bond between Cytochrome b and the Iron-Sulfur Protein

Xiao

2000

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To study the essentiality of head domain movement of the Rieske iron-sulfur protein (ISP) during bc 1 catalysis, Rhodobacter sphaeroides mutants expressing Histagged cytochrome bc 1 complexes with three pairs of cysteines engineered (one cysteine each) on the interface between cytochrome b and ISP, A185C(cytb)/ K70C(ISP), I326C(cytb)/G165C(ISP), and T386C(cytb)/ K164C(ISP), were generated and characterized. Formation of an intersubunit disulfide bond between cytochrome b and ISP is detected in membrane (intracytoplasmic membrane and air-aged chromatophore), and purified bc 1 complex was prepared from the A185C(cytb)/K70C(ISP) mutant cells. Formation of the intersubunit disulfide bond in this cysteine pair mutant complex is concurrent with the loss of its bc 1 activity. Reduction of this disulfide bond by ␤-mercaptoethanol restores activity, indicating that mobility of the head domain of ISP is functionally important in the cytochrome bc 1 complex. The rate of intramolecular electron transfer, between 2Fe2S and heme c 1 , in the A185C(cytb)/K70C(ISP) mutant complex is much lower than that in the wild type or in their respective single cysteine mutant complexes, indicating that formation of an intersubunit disulfide bond between cytochrome b and ISP arrests the head domain of ISP in the "fixed state" position, which is too far for electron transfer to heme c 1 .The cytochrome bc 1 complex (also known as ubiquinol-cytochrome c reductase or complex III) is an essential segment of the energy-conserving electron transfer chains of mitochondria and many respiratory and photosynthetic bacteria (1). This complex catalyzes electron transfer from ubiquinol to cytochrome c and concomitantly translocates protons across the membrane to generate a membrane potential and pH gradient for ATP synthesis. Recently the cytochrome bc 1 complexes from bovine (2, 3) and chicken (4) heart mitochondria, which contain 11 nonidentical protein subunits, were crystallized, and their structures were solved at 2.9 Å resolution. The structural information obtained not only answered a number of questions concerning the arrangement of the redox centers, transmembrane helices, and inhibitor binding sites but also suggested movement of the head domain of the iron-sulfur protein (ISP) 1 during bc 1 catalysis. This suggestion arose from observation of a particularly low electron density area in the intermembrane space portion of the complex, where the extramembrane domains of ISP and cytochrome c 1 reside (2). This movement hypothesis was further supported by the observation of various positions for 2Fe2S in the different crystal forms (3, 4) and in complexes loaded with different inhibitors (4, 5).In tetragonal I4 1 22 crystals of native oxidized bovine cytochrome bc 1 complex, the position of the 2Fe2S cluster is 27 Å from heme b L and 31 Å from heme c 1 (the "fixed state" position) (2, 5). Binding of stigmatellin or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole enhances the electron density of the anomalous scattering peak of 2Fe2S, suggesting that...

“…The selection of these three residues was based on the three-dimensional structure of the four-subunit cytochrome bc 1 complex of R. sphaeroides ( Fig. 2A) constructed by using coordinates from bovine cytochromes b and c 1 , ISP, and subunit VII (31). The distances between the symmetry pairs of aromatic residues Phe-195, Tyr-199, or Phe-203 are 4.5, 7.7, and 6.8 Å (Fig.…”

Section: Involvement Of the Phe-195 Of Cytochrome B In Electronmentioning

confidence: 99%

Evidence for Electron Equilibrium between the Two Hemes bL in the Dimeric Cytochrome bc1 Complex

Gong

Xia

et al. 2005

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The cytochrome bc 1 complex (also known as ubiquinol-cytochrome c reductase or Complex III) is an essential segment of the electron transfer chains of mitochondria and many respiratory and photosynthetic bacteria (1). It catalyzes electron transfer from ubiquinol to cytochrome c with concomitant translocation of protons across the membrane to generate a proton gradient and membrane potential for ATP synthesis. The "proton-motive Q-cycle" is the most favored mechanism for electron and proton transfer in this complex (2, 3). The central feature of the Q-cycle mechanism is the bifurcation of electrons from ubiquinol at the Qo site. The first electron of ubiquinol is transferred to the "high potential chain," consisting of the Rieske [2Fe-2S] cluster, housed in the iron-sulfur protein (ISP), 1 and heme c 1 , housed in cytochrome c 1 . Then the second electron of ubiquinol is passed through the "low potential chain" consisting of heme b L and heme b H , both housed in the cytochrome b subunit.Recently, mitochondrial cytochrome bc 1 complexes from beef (4, 5), chicken (6), and yeast (7) were crystallized, and their three-dimensional structures were determined. The structural information not only supports the Q-cycle mechanism but also suggests the complex functioning as a dimer. This suggestion stems from the following structural data (4 -7): (i) the intertwining of ISPs in the two bc 1 monomers such that the head domain of ISP in one monomer is physically close to and interacting with the cytochrome b and cytochrome c 1 in the 2-fold symmetry-related other monomer; (ii) the presence of two apparently non-communicating cavities in the dimeric complex, each connecting the Qo pocket of one monomer to the Qi pocket of the other; and (iii) the distance between the Fe atoms of the two hemes b L is only 21 Å, which is approximately the same as that between heme b L and b H in one monomer (Fig. 1). The short distance between the two hemes b L and the presence of several aromatic amino acid residues at the interface of the two cytochrome b proteins has promoted investigator to speculate the existence of electron transfer or equilibrating between the two hemes b L (8 -10). Recently the existence of an intertwined dimer in solution was confirmed (11) in the Rhodobacter sphaeroides bc 1 complex through the formation of a four-subunit (two ISPs and two cytochrome bs) adduct by two inter-subunit disulfide bonds between two engineered cysteine pairs: one at cytochrome b and the head domain of ISP and the other at cytochrome b and the tail domain of ISP. However, evidence for inter-monomer b L -b L electron transfer during bc 1 catalysis is still missing, due to the lack of a suitable assay method.It has been reported that during electron transfer through the bc 1 complex, superoxide anion (O 2 . ) is produced (12-18).This results from a leakage of the second electron of ubiquinol, from the low potential chain of the Q cycle electron transfer pathway, to interact with molecular oxygen. The electron-leaking site is thought to be lo...