In higher plants, cellulose is synthesized at the plasma membrane by the cellulose synthase (CESA) complex. The catalytic core of the complex is believed to be composed of three types of CESA subunits. Indirect evidence suggests that the complex associated with primary wall cellulose deposition consists of CESA1, -3, and -6 in Arabidopsis thaliana. However, phenotypes associated with mutations in two of these genes, CESA1 and -6, suggest unequal contribution by the different CESAs to overall enzymatic activity of the complex. We present evidence that the primary complex requires three unique types of components, CESA1-, CESA3-, and CESA6-related, for activity. Removal of any of these components results in gametophytic lethality due to pollen defects, demonstrating that primary-wall cellulose synthesis is necessary for pollen development. We also show that the CESA6-related CESAs are partially functionally redundant.gametophytic lethal ͉ isoforms ͉ pollen ͉ cellulose synthesis ͉ mutant

This review describes the recent knowledge about tightly bound lipids in membrane protein structures and deduces general principles of the binding interactions. Bound lipids are grouped in annular, nonannular, and integral protein lipids. The importance of lipid binding for vertical positioning and tight integration of proteins in the membrane, for assembly and stabilization of oligomeric and multisubunit complexes, for supercomplexes, as well as their functional roles are pointed out. Lipid binding is stabilized by multiple noncovalent interactions from protein residues to lipid head groups and hydrophobic tails. Based on analysis of lipids with refined head groups in membrane protein structures, distinct motifs were identified for stabilizing interactions between the phosphodiester moieties and side chains of amino acid residues. Differences between binding at the electropositive and electronegative membrane side, as well as a preferential binding to the latter, are observed. A first attempt to identify lipid head group specific binding motifs is made. A newly identified cardiolipin binding site in the yeast cytochrome bc(1) complex is described. Assignment of unsaturated lipid chains and evolutionary aspects of lipid binding are discussed.

Bifurcated electron transfer during ubiquinol oxidation is the key reaction of cytochrome bc 1 complex catalysis. Binding of the competitive inhibitor 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole to the Q o site of the cytochrome bc 1 complex from Saccharomyces cerevisiae was analyzed by x-ray crystallography. This alkylhydroxydioxobenzothiazole is bound in its ionized form as evident from the crystal structure and confirmed by spectroscopic analysis, consistent with a measured pK a ‫؍‬ 6.1 of the hydroxy group in detergent micelles. Stabilizing forces for the hydroxyquinone anion inhibitor include a polarized hydrogen bond to the iron-sulfur cluster ligand His 181 and on-edge interactions via weak hydrogen bonds with cytochrome b residue Tyr 279 . The hydroxy group of the latter contributes to stabilization of the Rieske protein in the b-position by donating a hydrogen bond. The reported pH dependence of inhibition with lower efficacy at alkaline pH is attributed to the protonation state of His 181 with a pK a of 7.5. Glu 272 , a proposed primary ligand and proton acceptor of ubiquinol, is not bound to the carbonyl group of the hydroxydioxobenzothiazole ring but is rotated out of the binding pocket toward the heme b L propionate A, to which it is hydrogen-bonded via a single water molecule. The observed hydrogen bonding pattern provides experimental evidence for the previously proposed proton exit pathway involving the heme propionate and a chain of water molecules. Binding of the alkyl-6-hydroxy-4,7-dioxobenzothiazole is discussed as resembling an intermediate step of ubiquinol oxidation, supporting a single occupancy model at the Q o site.Ubiquinol:cytochrome c oxidoreductase (cytochrome bc 1 complex, EC 1.10.2.2 (bc 1 complex)) is a multisubunit membrane protein complex, which is one of the fundamental components of respiratory and photosynthetic electron transfer chains. The enzyme catalyzes electron transfer from ubiquinol to cytochrome c and couples this process to electrogenic translocation of protons across the membrane (1, 2). Each functional unit of the homodimeric complex consists of three catalytic subunits: cytochrome b with two b type hemes, cytochrome c 1 with one c type heme, and the Rieske protein containing a [2Fe-2S] cluster. Mitochondrial bc 1 complexes contain up to eight additional subunits. Structures of vertebrate and yeast bc 1 complexes were determined, providing a breakthrough in understanding the enzyme mechanism and structure-function relationships within the enzyme (3-7). The 2.3-Å resolution structure from the yeast Saccharomyces cerevisiae has the highest resolution available so far. It allowed a detailed description of substrate and inhibitor binding sites, elucidating parts of the enzyme mechanism and suggesting pathways for proton transfer.The mechanism of the enzyme known as the protonmotive Q cycle (8) involves separate catalytic sites for ubiquinol oxidation (Q o site) and ubiquinone reduction (Q i site). Protons are taken up from the matrix side when ubiquinone is reduced a...

The cytochrome bc 1 complex catalyzes electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which electron transfer is linked to proton translocation across the inner mitochondrial membrane. In the Q cycle mechanism proton translocation is the net result of topographically segregated reduction of quinone and reoxidation of quinol on opposite sides of the membrane, with protons being carried across the membrane as hydrogens on the quinol. The linkage of proton chemistry to electron transfer during quinol oxidation and quinone reduction requires pathways for moving protons to and from the aqueous phase and the hydrophobic environment in which the quinol and quinone redox reactions occur. Crystal structures of the mitochondrial cytochrome bc 1 complexes in various conformations allow insight into possible proton conduction pathways. In this review we discuss pathways for proton conduction linked to ubiquinone redox reactions with particular reference to recently determined structures of the yeast bc 1 complex.

Atovaquone is a substituted 2-hydroxynaphthoquinone that is used therapeutically to treat Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis. It is thought to act on these organisms by inhibiting the cytochrome bc 1 complex. We have examined the interaction of atovaquone with the bc 1 complex isolated from Saccharomyces cerevisiae, a surrogate, nonpathogenic fungus. Atovaquone inhibits the bc 1 complex competitively with apparent K i ‫؍‬ 9 nM, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center. These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc 1 complex, where it interacts with the Rieske iron-sulfur protein. A computed energy-minimized structure for atovaquone liganded to the yeast bc 1 complex suggests that a phenylalanine at position 275 of cytochrome b in the bovine bc 1 complex, as opposed to leucine at the equivalent position in the yeast enzyme, is responsible for the decreased sensitivity of the bovine bc 1 complex (K i ‫؍‬ 80 nM) to atovaquone. When a L275F mutation was introduced into the yeast cytochrome b, the sensitivity of the yeast enzyme to atovaquone decreased (K i ‫؍‬ 100 nM) with no loss in activity, confirming that the L275F exchange contributes to the differential sensitivity of these two species to atovaquone. These results provide the first molecular description of how atovaquone binds to the bc 1 complex and explain the differential inhibition of the fungal versus mammalian enzymes.