Quinone Reduction Site Research Articles

Monitoring the redox and protonation dependent contributions of cardiolipin in electrochemically induced FTIR difference spectra of the cytochrome bc1 complex from yeast

Biochemical studies have shown that cardiolipin is essential for the integrity and activity of the cytochrome bc 1 complex and many other membrane proteins. Recently the direct involvement of a bound cardiolipin molecule (CL) for proton uptake at center N, the site of quinone reduction, was suggested on the basis of a crystallographic study. In the study presented here, we probe the low frequency infrared spectroscopy region as a technique suitable to detect the involvement of the lipids in redox induced reactions of the protein. First the individual infrared spectroscopic features of lipids, typically present in the yeast membrane, have been monitored for different pH values in micelles and vesicles. The p K a values for cardiolipin molecule have been observed at 4.7 ± 0.3 and 7.9 ± 1.3, respectively. Lipid contributions in the electrochemically induced FTIR spectra of the bc 1 complex from yeast have been identified by comparing the spectra of the as isolated form, with samples where the lipids were digested by lipase-A 2. Overall, a noteworthy perturbation in the spectral region typical for the protein backbone can be reported. Interestingly, signals at 1159, 1113, 1039 and 980 cm − 1 have shifted, indicating the perturbation of the protonation state of cardiolipin coupled to the reduction of the hemes. Additional shifts are found and are proposed to reflect lipids reorganizing due to a change in their direct environment upon the redox reaction of the hemes. In addition a small shift in the alpha band from 559 to 556 nm can be seen after lipid depletion, reflecting the interaction with heme b H and heme c. Thus, our work highlights the role of lipids in enzyme reactivity and structure.

S2/6 The unusual configuration of the quinone reduction site of the cytochrome b6f complex

S2/6 The unusual configuration of the quinone reduction site of the cytochrome b6f complex

Inhibitor Complexed Structures of the Cytochrome bc1 from the Photosynthetic Bacterium Rhodobacter Sphaeroides

The cytochrome bc1 complex (bc1) is a major contributor to the proton motive force across the membrane by coupling electron transfer to proton translocation. The crystal structures of wild type and mutant bc1 complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides (Rsbc1), stabilized with the quinol oxidation (QP) site inhibitor stigmatellin alone or in combination of with the quinone reduction (QN) site inhibitor antimycin, were determined. The high quality electron density permitted assignments of a new metal‐binding site to the cytochrome c1 subunit and a number of lipid and detergent molecules. Structural differences between Rsbc1 and its mitochondrial counterparts are mostly extra membranous and provide a basis for understanding the function of the predominantly longer sequences in the bacterial subunits. Functional implications for the bc1 complex are derived from analyses of 10 independent molecules in various crystal forms and from comparisons with mitochondrial complexes.

Inhibitor-complexed Structures of the Cytochrome bc1 from the Photosynthetic Bacterium Rhodobacter sphaeroides

The cytochrome bc(1) complex (bc(1)) is a major contributor to the proton motive force across the membrane by coupling electron transfer to proton translocation. The crystal structures of wild type and mutant bc(1) complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides (Rsbc(1)), stabilized with the quinol oxidation (Q(P)) site inhibitor stigmatellin alone or in combination with the quinone reduction (Q(N)) site inhibitor antimycin, were determined. The high quality electron density permitted assignments of a new metal-binding site to the cytochrome c(1) subunit and a number of lipid and detergent molecules. Structural differences between Rsbc(1) and its mitochondrial counterparts are mostly extra membranous and provide a basis for understanding the function of the predominantly longer sequences in the bacterial subunits. Functional implications for the bc(1) complex are derived from analyses of 10 independent molecules in various crystal forms and from comparisons with mitochondrial complexes.

Differential efficacy of inhibition of mitochondrial and bacterial cytochrome bc1 complexes by center N inhibitors antimycin, ilicicolin H and funiculosin

We have compared the efficacy of inhibition of the cytochrome bc 1 complexes from yeast and bovine heart mitochondria and Paracoccus denitrificans by antimycin, ilicicolin H, and funiculosin, three inhibitors that act at the quinone reduction site at center N of the enzyme. Although the three inhibitors have some structural features in common, they differ significantly in their patterns of inhibition. Also, while the overall folding pattern of cytochrome b around center N is similar in the enzymes from the three species, amino acid sequence differences create sufficient structural differences so that there are striking differences in the inhibitors binding to the three enzymes. Antimycin is the most tightly bound of the three inhibitors, and binds stoichiometrically to the isolated enzymes from all three species under the cytochrome c reductase assay conditions. Ilicicolin H also binds stoichiometrically to the yeast enzyme, but binds approximately 2 orders of magnitude less tightly to the bovine enzyme and is essentially non-inhibitory to the Paracoccus enzyme. Funiculosin on the other hand inhibits the yeast and bovine enzymes similarly, with IC 50 ∼ 10 nM, while the IC 50 for the Paracoccus enzyme is more than 10-fold higher. Similar differences in inhibitor efficacy were noted in bc 1 complexes from yeast mutants with single amino acid substitutions at the center N site, although the binding affinity of quinone and quinol substrates were not perturbed to a degree that impaired catalytic function in the variant enzymes. These results reveal a high degree of specificity in the determinants of ligand-binding at center N, accompanied by sufficient structural plasticity for substrate binding as to not compromise center N function. The results also demonstrate that, in principle, it should be possible to design novel inhibitors targeted toward center N of the bc 1 complex with appropriate species selectivity to allow their use as drugs against pathogenic fungi and parasites.

Hydrogen Bonds between Nitrogen Donors and the Semiquinone in the Qi-site of the bc1 Complex

The ubisemiquinone stabilized at the Qi-site of the bc1 complex of Rhodobacter sphaeroides forms a hydrogen bond with a nitrogen from the local protein environment, tentatively identified as ring N from His-217. The interactions of 14N and 15N have been studied by X-band (approximately 9.7 GHz) and S-band (3.4 GHz) pulsed EPR spectroscopy. The application of S-band spectroscopy has allowed us to determine the complete nuclear quadrupole tensor of the 14N involved in H-bond formation and to assign it unambiguously to the Nepsilon of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and Ndelta in other quinone-processing sites. The experiments with 15N showed that the Nepsilon of His-217 was the only nitrogen carrying any considerable unpaired spin density in the ubiquinone environment, and allowed calculation of the isotropic and anisotropic couplings with the Nepsilon of His-217. From these data, we could estimate the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and the distance from the nitrogen to the carbonyl oxygen of 2.38+/-0.13A. The hyperfine coupling of other protein nitrogens with semiquinone is <0.1 MHz. This did not exclude the nitrogen of the Asn-221 as a possible hydrogen bond donor to the methoxy oxygen of the semiquinone. A mechanistic role for this residue is supported by kinetic experiments with mutant strains N221T, N221H, N221I, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover.

Structural basis for the mechanism of electron bifurcation at the quinol oxidation site of the cytochrome bc 1 complex

At the heart of the Q cycle hypothesis, the cytochrome bc1 complex (bc1) is required to separate the two electrons from a quinol molecule at the quinol oxidation site. Recent studies have brought to light an intricate mechanism for this bifurcated electron transfer. A survey of the protein data bank shows 30 entries for the structures of bc1 and the homologous b6 f complex. These structures provide considerable insights into the structural organization of mitochondrial, bacterial, and plant enzymes. Crystallographic binding studies of bc1 with either quinone reduction (QN) and/or quinol oxidation (QP) site inhibitors offer atomic details on how these compounds interact with residues at their respective sites. Most importantly, the different locations and apparent flexibility observed in crystals for the extrinsic domain of the iron-sulfur protein (ISP) subunit suggest a mechanism for electron bifurcation at the QP site. Analyses of various inhibitor-bound structures revealed two classes of QP site inhibitors: Pm inhibitors that promote ISP mobility and Pf inhibitors that favor the fixation of the ISP conformation. Those analyses also shed light on a possible process by which the ISP motion switch is controlled. The first phase reduction of ISP is shown to be comparable to the reduction of the bL heme by pre-steady state kinetic analysis, whereas the second phase reduction of ISP share similar kinetics with the reduction of the bH heme. The reduction of cyt c1 is measured much slower, indicating that the reduced ISP remains bound at the QP site until the reduced heme bL is oxidized by the heme bH and supporting the existence of a control mechanism for the ISP motion switch.

Effect of Inhibition of the bc1 Complex on Gene Expression Profile in Yeast

Because the respiratory chain is the major site of oxidation of the reduced equivalents and of energy production in aerobic cells, its inhibition has severe impact on the cells. Communication pathways from the respiratory chain are required to allow the cell to sense the defect and respond to it. In this work, we studied changes in gene expression induced by the treatment of yeast cells with myxothiazol, an inhibitor of the bc(1) complex, an enzyme of the respiratory chain. The pattern and time-course expression of the genes resemble those of the environmental stress response, a common gene expression program induced by sudden changes in the environment. In addition, the changes were, for most of the genes, mediated through the transcription factors Msn2/4, which play a central role in the cellular response to these stresses. By using a mutant with a myxothiazol-resistant bc(1) complex, we showed that the changes of expression of the majority of the genes was caused by the inhibition of the bc(1) complex but that other stresses might be involved. The expression pattern of CTT1, coding for a cytoplasmic catalase, was further studied. The expression of this gene was largely dependent on Msn2/4 and the inhibition of the cytochrome bc(1). Addition of oxidants of NADH was found to decrease the expression of CTT1 induced by myxothiazol treatment, suggesting that the accumulation of NADH caused by the inhibition of the respiratory chain may be involved in the signaling pathway from the mitochondria to the transcription factor.

Cardiolipin Is Essential for Organization of Complexes III and IV into a Supercomplex in Intact Yeast Mitochondria

Digitonin extracts of mitochondria from cardiolipin-containing (wild type) and cardiolipin-lacking (crd1Delta mutant) Saccharomyces cerevisiae subjected to colorless native polyacrylamide gel electrophoresis in the presence of 0.003% digitonin displayed a supercomplex composed of homodimers of complexes III and IV in the former case but only the individual homodimers in the latter case. To avoid treatment with any detergent or dye, we compared organization of the respiratory chain in intact mitochondria from wild type and cardiolipin-lacking cells by using a functional analysis developed previously for the study of the organization of the respiratory chain of S. cerevisiae (Boumans, H., Grivell, L. A., and Berden, J. A. (1998) J. Biol. Chem. 273, 4872-4877). Dependence of the kinetics of NADH oxidation via complexes III, IV, and cytochrome c on the concentration of the complex III-specific inhibitor antimycin A was studied. A linear relationship between respiratory activity and saturation of complex III with antimycin A was obtained for wild type mitochondria consistent with single functional unit kinetics of the respiratory chain. Under the same conditions, cardiolipin-lacking mitochondria displayed a hyperbolic relationship indicating cytochrome c pool behavior. No release of cytochrome c from cardiolipin-lacking mitochondria or mitoplasts under our standard experimental conditions was detected. Identical cytochrome c pool behavior was observed for both wild type and cardiolipin-lacking mitochondria in the presence of a chaotropic agent, which disrupts the interaction between respiratory complexes. The results demonstrate that cardiolipin is essential for association of complexes III and IV into a supercomplex in intact yeast mitochondria.

Binding of the Respiratory Chain Inhibitor Antimycin to the Mitochondrial bc1 Complex: A New Crystal Structure Reveals an Altered Intramolecular Hydrogen-bonding Pattern

Antimycin A (antimycin), one of the first known and most potent inhibitors of the mitochondrial respiratory chain, binds to the quinone reduction site of the cytochrome bc1 complex. Structure-activity relationship studies have shown that the N-formylamino-salicyl-amide group is responsible for most of the binding specificity, and suggested that a low pKa for the phenolic OH group and an intramolecular H-bond between that OH and the carbonyl O of the salicylamide linkage are important. Two previous X-ray structures of antimycin bound to vertebrate bc1 complex gave conflicting results. A new structure reported here of the bovine mitochondrial bc1 complex at 2.28 A resolution with antimycin bound, allows us for the first time to reliably describe the binding of antimycin and shows that the intramolecular hydrogen bond described in solution and in the small-molecule structure is replaced by one involving the NH rather than carbonyl O of the amide linkage, with rotation of the amide group relative to the aromatic ring. The phenolic OH and formylamino N form H-bonds with conserved Asp228 of cytochrome b, and the formylamino O H-bonds via a water molecule to Lys227. A strong density, the right size and shape for a diatomic molecule is found between the other side of the dilactone ring and the alphaA helix.

Rhodoquinone reaction site of mitochondrial complex I, in parasitic helminth, Ascaris suum

The components and organization of the respiratory chain in helminth mitochondria vary remarkably depending upon the stage of the life cycle. Mitochondrial complex I in the parasitic helminth Ascaris suum uses ubiquinone-9 (UQ 9) and rhodoquinone-9 (RQ 9) under aerobic and anaerobic conditions, respectively. In this study, we investigated structural features of the quinone reduction site of A. suum complex I using a series of quinazoline-type inhibitors and also by the kinetic analysis of rhodoquinone-2 (RQ 2) and ubiquinone-2 (UQ 2) reduction. Structure–activity profiles of the inhibition by quinazolines were comparable, but not completely identical, between NADH-RQ 2 and NADH-UQ 2 oxidoreductase activities. However, the inhibitory mechanism of quinazolines was competitive and partially competitive against RQ 2 and UQ 2, respectively. The pH profiles of both activities differed remarkably; NADH-RQ 2 oxidoreductase activity showed an optimum pH at 7.6, whereas NADH-UQ 2 oxidoreductase activity showed two optima pH at 6.4 and 7.2. Our results indicate that although A. suum complex I uses both RQ 2 and UQ 2 as an electron acceptor, the manner of reaction (or binding) of the two quinones differs.

Reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes

Hydrogenase and fumarate reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed fumarate reduction by H2 which generated an electrical proton potential (Δψ = 0.19 V, negative inside) in the same direction as that generated by fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Δψ and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without fumarate reductase. Proteoliposomes containing menaquinone and fumarate reductase with or without hydrogenase catalyzed fumarate reduction by DMNH2 which did not generate a Δψ. Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Δψ of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Δψ generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by fumarate. Δψ generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.

Reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes.

Hydrogenase and fumarate reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed fumarate reduction by H2 which generated an electrical proton potential (Delta(psi) = 0.19 V, negative inside) in the same direction as that generated by fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Delta(psi) and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without fumarate reductase. Proteoliposomes containing menaquinone and fumarate reductase with or without hydrogenase catalyzed fumarate reduction by DMNH2 which did not generate a Delta(psi). Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Delta(psi) of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Delta(psi) generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by fumarate. Delta(psi) generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.

Preliminary X-ray crystallographic analysis ofMntC, a periplasmic manganese binding component of a Mn transporter from Synechocystis sp. PCC 6803

Photosystem II contains the site of photosynthetic electron transfer initiation, water oxidation and quinone reduction in higher plants, algae and cyanobacteria. This complex contains a reaction center (RCII), antenna proteins and additional proteins with accessory or regulatory roles. In order to elucidate the biochemical and biophysical characteristics of the entire system, it is necessary to obtain information on the three-dimensional structures of all of the protein constituents. Our laboratory is involved in the determination of the structure of a number of PSII components. 1) We have crystallized the RCII complex from higher plants (monomeric and dimeric forms). Monomeric oxygen-evolving RCII from higher plants was crystallized in nine different detergent mixtures. We have used a variety of methodologies to probe the interaction between these detergents and the RCII complex. Recent results with cyanobacteria will also be shown. 2) We have solved the structure of C-phycocyanin, a PSII phycobilisome antenna protein from Synechococcus vulcanus at 2.5A (PDB code 1I7Y). We have identified structural characteristics leading to thermal stability in phycobilisomes, by comparisons with other phycocyanins of known structure. 3) We have obtained crystals diffracting to 3.2A, and begun structure determination, of the MntC protein from Synechocystis sp. PCC 6803, which is one of the proteins required for uptake and transport of manganese ions needed in PSII. 4) We have obtained microcrystals of dimers of spinach OEC manganese stabilizing protein (33 kDa protein). We have studied the behavior of this protein in vitro, and the results have lead to proposals on the function of this protein in vivo.

Toward a characterization of the connecting module of complex I.

Complex I [NADH-ubiquinone oxidoreductase (complex I, EC 1.6.5.3)] couples electron transfer between NADH and ubiquinone to proton transport across the bacterial cytoplasmic membrane and the mitochondrial inner membrane. This sophisticated enzyme consists of three specialized modules: (1) a hydrophilic NADH-oxidizing module that constitutes the input machinery of the enzyme; (2) a hydrophobic module that anchors the enzyme in the membrane and must take part in proton transport; and (3) a connecting domain that links the two previous modules. Using the complex I of Rhodobacter capsulatus, we developed a genetic study of the structure and function of the connecting module. In the present review, we put together the salient results of these studies, with recent reports of the literature, to try and elucidate the structure of the connecting module and its potential role in the coupling process between electron and proton flux within complex I. From this overview, we conclude that the NUOB-NUOD dimer of the connecting module and a hydrophobic subunit such as NUOH must share a quinone-reduction site. The function of this site in the mechanism of complex I is discussed.

Structure of the avian mitochondrial cytochrome bc1 complex.

There are now four structures of vertebrate mitochondrial bc1 complexes available in the protein databases and structures from yeast and bacterial sources are expected soon. This review summarizes the new information with emphasis on the avian cytochrome bc1 complex (PDB entries 1BCC and 3BCC). The Rieske iron-sulfur protein is mobile and this has been proposed to be important for catalysis. The binding sites for quinone have been located based on structures containing inhibitors and, in the case of the quinone reduction site Qi, the quinone itself.

Steps toward constructing a cytochrome b6 f complex in the purple bacterium Rhodobacter sphaeroides: an example of the structural plasticity of a membrane cytochrome.

We have modified the cytochrome b subunit of the cytochrome bc1 complex from the purple bacterium Rhodobacter sphaeroides to introduce two distinctive features of cytochrome b6 f complexes. In the first one, we have split cyt b into two polypeptides thus mimicking the organization of cyt b6 and subunit IV in the b6 f complexes. In the second, an extra residue was added between His198 and Phe199, thus extending the span between the histidine ligands for the two b-hemes in helix D. The properties of the mutant strains were determined using thermodynamic and kinetic analysis. The two mutant enzymes were assembled and functioned so as to allow the photosynthetic growth of the mutant strains. For the split enzyme, we show that two independently translated fragments of cyt b are inserted in the membrane. Our results indicate a decrease in the stability of the semiquinone formed at the quinone reduction (Qi) site in this mutant. This property, characteristic for b6 f complexes, indicates the functional importance of the connecting span between helices D and E. The presence of the inserted threonine in helix D modified the spectrum and redox potential of the bL-heme, shifting the potential difference between the two b-hemes from 140 mV in the wild-type to 55 mV in the mutant strain. This change in the driving force of electron transfer through the membrane was reflected in an inability of the mutant strain to accumulate a large transmembrane electrical potential on successive flashes.

Characterization of the ubiquinone reduction site of mitochondrial complex I using bulky synthetic ubiquinones.

A wide variety of alkyl derivatives of Q2 (6-geranyl-2, 3-dimethoxy-5-methyl-1,4-benzoquinone) and DB (6-n-decyl-2, 3-dimethoxy-5-methyl-1,4-benzoquinone), in which methoxy groups of the 2- and/or 3-positions of the quinone ring were replaced by other bulky alkoxy groups from ethoxy to butoxy, were prepared by novel synthetic procedures. Electron-accepting activities of the bulky quinones were investigated with bovine heart mitochondrial complex I and its counterpart of Paracoccus denitrificans(NDH-1) to elucidate structural and functional features of the quinone reduction site of the enzymes. The bulky quinone analogues served as sufficient electron acceptors from the physiological quinone reduction site of bovine complex I. Considering the very poor activities of even the ethoxy derivatives as substrates for other respiratory enzymes such as mitochondrial complexes II and III [He, D. Y., Gu, L. Q., Yu, L., and Yu, C. A. (1994) Biochemistry 33, 880-884], this result indicated that the quinone reduction site of bovine complex I is spacious enough to accommodate bulky exogenous substrates. In contrast to bovine complex I, bulky quinone analogues served as poor electron acceptors with Paracoccus NDH-1. These observations indicated that bovine complex I recognizes the substrate structure with poor specificity. The substituent effects in the 2- and 3-positions of the quinone ring on the electron-transfer activity with bovine complex I differed significantly between Q2 and DB series despite having the same total number of carbon atoms in the side chain. The inhibitory effect involving Q2 due to its geranyl side chain was markedly diminished by structural modifications of the quinone ring moiety. These findings indicate that the side chain plays a specific role in the redox reaction and that the quinone ring and side-chain moieties contribute interdependently to binding interaction. Moreover, structural dependency of the proton-pumping activity of the quinone analogues was comparable to that of the electron-transfer activity with bovine complex I, indicating that the mechanism of redox-driven proton-pumping does not differ depending upon the substrate structure.

Inhibition of cytochrome b563-oxidation by triorganotins in spinach chloroplasts

The triorganotin compounds triphenyltin chloride and tributyltin chloride have been known as inhibitors of the transmembrane proton channel forming F0-domain of ATPases at micromolar concentrations. We show that these compounds at higher concentrations (10–100 µM) also inhibit uncoupled electron transport in chloroplasts within the low potential chain of the cytochrome bf complex. They cause high levels of transiently reduced cytochrome b563 as they decelerate the reoxidation process in flash illuminated chloroplasts. At the same time they slow down the flash induced slow electrogenic step generated at the cytochrome bf complex. The inhibitory effect of triphenyltin chloride on cytochrome b563 turnover in chloroplasts is comparable to that of the Qn-inhibitor MOA-stilbene, with even less side effects on the high potential chain. Studies on the isolated bf complex suggest different binding sites for triorganotins and the quinone analogue type Qn-inhibitors. The results are interpreted within the framework of the modified Q-cycle model by a putative organotin sensitive proton translocating site which enables proton transfer from the outer aqueous face of the membrane to the hydrophobic quinone reduction site within the complex. Hence, cytochrome b563 oxidation and plastoquinone reduction may be inhibited as a consequence of proton transfer being suppressed by triorganotins. In analogy, the previously described inhibitory effect of Val/K+ at the n-side of the cytochrome bf complex [Klughammer and Schreiber (1993) FEBS 336: 491–495] may be rationalised by binding of the cyclic depsipeptide at the entrance of the proton path to the Qn-site.

Analysis of revertants from respiratory deficient mutants within the center N of cytochrome b in Saccharomyces cerevisiae

Four modified cytochrome b's carrying mononucleotide substitutions affecting center N residues were analysed. The mutant carrying a G33D change does not incorporate heme into the apocytochrome b and fails to grow on non-fermentable carbon sources. Out of 85 genetically independent revertants derived from this mutant, 82 were true back-mutants restoring the wild type sequence (D33G). The remaining three replaced the aspartic acid by an alanine (D33A) indicating that small size residues are best tolerated at this position which is consistent with the perfect conservation of the G33 during evolution. This glycine may be of crucial importance for helix packing around the hemes. The replacement of methionine at position 221 by lysine (M221K.) produced a non-functional cytochrome b [(1993) J. Biol. Chem. 268,15626-15632]. Non-native revertants replacing the lysine 221 by glutamic acid (K221E) or glutamine (K221Q) expressed a selective resistance to antimycin and antimycin derivatives having a modified dilactone ring moiety. Cytochrome b residues in 33 and in 221 seemed to contribute to the quinone reduction (QN) site of the cytochrome bc1 complex. Possible intramolecular interactions between the N-tenninal region and the loop connecting helices IV and V of cytochrome b are proposed.