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

Abstract

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...

Electronic Connection Between the Quinone and CytochromecRedox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling

“…The two monomeric units assemble so that the FeS head domain interacting with cytochrome b and cytochrome c 1 of one monomer has its membranous anchor associated with the other monomer. Dimeric nature of cytochrome bc 1 was further confirmed in biochemical studies (270). Interestingly, two hemes b L in the dimer are in a close distance (14 Å), implicating that electronic connection between monomers via heme b L -b L electron transfer is possible (FIGURE 5B).…”

Simultaneous reduction of iron–sulfur protein and cytochrome b L during ubiquinol oxidation in cytochrome bc 1 complex

“…The iron-sulfur proteins (ISPs) in the two bc 1 monomers are intertwined with the head domain in one monomer interacting with cytochrome b and cytochrome c 1 of the other monomer. The intertwined dimer observed in the crystalline complex also exists in solution, which was confirmed (8) in the R. sphaeroides bc 1 complex through the formation of a four-subunit (two ISPs and two cytochrome bs) adduct by two intersubunit disulfide bonds between two engineered cysteine pairs: one pair links the ISP head domain to cytochrome b, and the other pair links the ISP tail domain to cytochrome b of another monomer. Two apparently noncommunicating cavities in the dimeric complex are presented: each connecting the Qp pocket of one monomer to the Qn pocket of the other.…”

Failure to Insert the Iron-Sulfur Cluster into the Rieske Iron-Sulfur Protein Impairs Both Center N and Center P of the Cytochrome bc 1 Complex

“…1A. The head domain of the iron-sulfur protein comes in close contact with heme b L of cytochrome b and cytochrome c 1 of one monomer, whereas the N-terminal and transmembrane domains of the protein are anchored in the other monomer, close to heme b H. Confirmation of an intertwined dimer in solution was provided when a stable bc 1 complex from Rhodobacter sphaeroides was generated carrying two intersubunit disulfide bonds, one between the head domain of the iron-sulfur protein and cytochrome b and the other between the tail domain of the ironsulfur protein and cytochrome b (21).…”