Quinone Analogue Inhibitors Research Articles

Increased Superoxide Production in the Cytochrome B6F Complex: A Function for the Enigmatic Chlorophyll-A

The structural basis for superoxide production in cytochrome bc complexes is relevant to understanding the mechanism of generation of deleterious reactive oxygen species and partition of electron transfer in the branched quinol oxidation pathway in cytochrome bc complexes. The specific rate of superoxide production, normalized to the the electron transfer rate, was determined for the yeast cytochrome bc1 complex (provided by B. L. Trumpower) and the cytochrome b6f complex from spinach thylakoid membranes and cyanobacteria. Although electron transfer rates were comparable in bc1 and b6f complexes, the specific rate of superoxide production was 10-20 fold greater in the b6f complex. Whereas antimycin A, a specific n-side quinone analogue inhibitor of the cytochrome bc1 complex, caused a large increase in the superoxide production rate of the bc1 complex, no comparable effect was found for NQNO, an n-side quinone analogue inhibitor in b6f, as defined by spectrophotometry and a crystal structure. These differences between bc1 and b6f complexes imply an increase in branching ratio for reduction by plasto-semiquinone of O2 to O2-, relative to reduction of heme bp for trans-membrane electron transfer. The change in branching ratio is ascribed to a longer semiquinone residence time in the p-side binding niche, due to steric restriction of the quinone binding site by the chlorophyll phytyl chain, as seen in a crystal structure. The presence of this phytyl chain can be seen to result in a smaller accessible volume for binding sites of a p-side quinone analogue inhibitor. The longer residence time of quinol/semiquinone would facilitate trans-membrane signaling, e.g., activation of n-side LHC kinase (NIH GM-38323).

An Anhydrous Proton Transfer Pathway in the Cytochrome B6F Complex

Cytochrome b6f, a hetero-oligomeric energy transducing membrane protein complex, catalyzes proton-coupled electron transfer reactions of the substrate quinone to generate as much as two-thirds of the total proton gradient used for ATP synthesis in oxygenic photosynthesis. Proton transfer pathways within b6f have remained unidentified due to limitations of crystallographic resolution1,2,3,4,5. using new crystallographic information based on a 2.70 A crystal structure of the native complex, and structures obtained in the presence of quinone analogue inhibitors, tridecyl-stigmatellin (TDS) and 2-nonyl-4-hydroxyquinoline-N-oxide (NQNO) (resolution 3.07 A and 3.25 A respectively) seen in close proximity to heme cn on the electrochemically negative (n) side of the b6f complex, an anhydrous proton uptake pathway is defined that delivers protons from the n-side aqueous phase to plastoquinone bound at heme cn. A hydrated channel is identified on the electrochemically positive (p) side of the complex, that may provide a route for proton exit to the p-side aqueous phase. These n- and p-side pathways contribute to the first complete description of quinone-mediated trans-membrane proton transfer.1. Kurisu et al. 2003, 2Stroebel et al. 2003, 3Yan et al. 2006, 4Yamashita et al. 2007, 5Baniulis et al. 2009View Large Image | View Hi-Res Image | Download PowerPoint Slide

The alternative complex III of Rhodothermus marinus and its structural and functional association with caa3 oxygen reductase

An alternative complex III (ACIII) is a respiratory complex with quinol:electron acceptor oxidoreductase activity. It is the only example of an enzyme performing complex III function that does not belong to bc 1 complex family. ACIII from Rhodothermus (R.) marinus was the first enzyme of this type to be isolated and characterized, and in this work we deepen its characterization. We addressed its interaction with quinol substrate and with the caa 3 oxygen reductase, whose coding gene cluster follows that of the ACIII. There is at least, one quinone binding site present in R. marinus ACIII as observed by fluorescence quenching titration of HQNO, a quinone analogue inhibitor. Furthermore, electrophoretic and spectroscopic evidences, taken together with mass spectrometry revealed a structural association between ACIII and caa 3 oxygen reductase. The association was also shown to be functional, since quinol:oxygen oxidoreductase activity was observed when the two isolated complexes were put together. This work is thus a step forward in the recognition of the structural and functional diversities of prokaryotic respiratory chains.

Biochemical and Spectroscopic Properties of Cyanide-Insensitive Quinol Oxidase from Gluconobacter oxydans

Cyanide-insensitive quinol oxidase (CioAB), a relative of cytochrome bd, has no spectroscopic features of hemes b(595) and d in the wild-type bacteria and is difficult to purify for detailed characterization. Here we studied enzymatic and spectroscopic properties of CioAB from the acetic acid bacterium Gluconobacter oxydans. Gluconobacter oxydans CioAB showed the K(m) value for ubiquinol-1 comparable to that of Escherichia coli cytochrome bd but it was more resistant to KCN and quinone-analogue inhibitors except piericidin A and LL-Z1272gamma. We obtained the spectroscopic evidence for the presence of hemes b(595) and d. Heme b(595) showed the alpha peak at 587 nm in the reduced state and a rhombic high-spin signal at g = 6.3 and 5.5 in the air-oxidized state. Heme d showed the alpha peak at 626 and 644 nm in the reduced and air-oxidized state, respectively, and an axial high-spin signal at g = 6.0 and low-spin signals at g = 2.63, 2.37 and 2.32. We found also a broad low-spin signal at g = 3.2, attributable to heme b(558). Further, we identified the presence of heme D by mass spectrometry. In conclusion, CioAB binds all three ham species present in cytochrome bd quinol oxidase.

Structure–Function of the Cytochrome b6f Complex†

The structure and function of the cytochrome b6f complex is considered in the context of recent crystal structures of the complex as an eight subunit, 220 kDa symmetric dimeric complex obtained from the thermophilic cyanobacterium, Mastigocladus laminosus, and the green alga, Chlamydomonas reinhardtii. A major problem confronted in crystallization of the cyanobacterial complex, proteolysis of three of the subunits, is discussed along with initial efforts to identify the protease. The evolution of these cytochrome complexes is illustrated by conservation of the hydrophobic heme-binding transmembrane domain of the cyt b polypeptide between b6f and bc1 complexes, and the rubredoxin-like membrane proximal domain of the Rieske [2Fe-2S] protein. Pathways of coupled electron and proton transfer are discussed in the framework of a modified Q cycle, in which the heme c(n), not found in the bc1 complex, but electronically tightly coupled to the heme b(n) of the b6f complex, is included. Crystal structures of the cyanobacterial complex with the quinone analogue inhibitors, NQNO or tridecyl-stigmatellin, show the latter to be ligands of heme c(n), implicating heme c(n) as an n-side plastoquinone reductase. Existing questions include (a) the details of the shuttle of: (i) the [2Fe-2S] protein between the membrane-bound PQH2 electron/H+ donor and the cytochrome f acceptor to complete the p-side electron transfer circuit; (ii) PQ/PQH2 between n- and p-sides of the complex across the intermonomer quinone exchange cavity, through the narrow portal connecting the cavity with the p-side [2Fe-2S] niche; (b) the role of the n-side of the b6f complex and heme c(n) in regulation of the relative rates of noncyclic and cyclic electron transfer. The likely presence of cyclic electron transport in the b6f complex, and of heme c(n) in the firmicute bc complex suggests the concept that hemes b(n)-c(n) define a branch point in bc complexes that can support electron transport pathways that differ in detail from the Q cycle supported by the bc1 complex.

Structure of the Cytochrome b6f Complex: Quinone Analogue Inhibitors as Ligands of Heme cn

A native structure of the cytochrome b 6 f complex with improved resolution was obtained from crystals of the complex grown in the presence of divalent cadmium. Two Cd 2+ binding sites with different occupancy were determined: (i) a higher affinity site, Cd1, which bridges His143 of cytochrome f and the acidic residue, Glu75, of cyt b 6; in addition, Cd1 is coordinated by 1–2 H 2O or 1–2 Cl −; (ii) a second site, Cd2, of lower affinity for which three identified ligands are Asp58 (subunit IV), Glu3 (PetG subunit) and Glu4 (PetM subunit). Binding sites of quinone analogue inhibitors were sought to map the pathway of transfer of the lipophilic quinone across the b 6 f complex and to define the function of the novel heme c n. Two sites were found for the chromone ring of the tridecyl-stigmatellin (TDS) quinone analogue inhibitor, one near the p-side [2Fe-2S] cluster. A second TDS site was found on the n-side of the complex facing the quinone exchange cavity as an axial ligand of heme c n. A similar binding site proximal to heme c n was found for the n-side inhibitor, NQNO. Binding of these inhibitors required their addition to the complex before lipid used to facilitate crystallization. The similar binding of NQNO and TDS as axial ligands to heme c n implies that this heme utilizes plastoquinone as a natural ligand, thus defining an electron transfer complex consisting of hemes b n, c n, and PQ, and the pathway of n-side reduction of the PQ pool. The NQNO binding site explains several effects associated with its inhibitory action: the negative shift in heme c n midpoint potential, the increased amplitude of light-induced heme b n reduction, and an altered EPR spectrum attributed to interaction between hemes c n and b n. A decreased extent of heme c n reduction by reduced ferredoxin in the presence of NQNO allows observation of the heme c n Soret band in a chemical difference spectrum.

Intraprotein transfer of the quinone analogue inhibitor 2,5-dibromo-3-methyl-6-isopropyl- p -benzoquinone in the cytochrome b 6 f complex

Details are presented of the structural analysis of the cytochrome b(6)f complex from the thermophilic cyanobacterium, Mastigocladus laminosus, in the presence of the electrochemically positive (p)-side quinone analogue inhibitor, 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB). One DBMIB binding site was found. This site is peripheral to the quinone binding space defined by the binding sites of other p-side inhibitors previously resolved in cytochrome bc(1)/b(6)f complexes. This high-affinity site resides in a p-side interfacial niche bounded by cytochrome f, subunit IV, and cytochrome b(6), is close (8 A) to the p-side heme b, but distant (19 A) from the [2Fe-2S] cluster. No significant electron density associated with the DBMIB was found elsewhere in the structure. However, the site at which DBMIB can inhibit light-induced redox turnover is within a few A of the [2Fe-2S] cluster, as shown by the absence of inhibition in mutants of Synechococcus sp. PCC 7002 at iron sulfur protein-Leu-111 near the cluster. The ability of a minimum amount of initially oxidized DBMIB to inhibit turnover of WT complex after a second light flash implies that there is a light-activated movement of DBMIB from the distal peripheral site to an inhibitory site proximal to the [2Fe-2S] cluster. Together with the necessary passage of quinone/quinol through the small Q(p) portal in the complex, it is seen that transmembrane traffic of quinone-like molecules through the core of cytochrome bc complexes can be labyrinthine.

Binding sites of lipophilic quinone and quinone analogue inhibitors in the cytochrome b6f complex of oxygenic photosynthesis

The main structural features of the cytochrome b6f complex, solved to 3.0–3.1 Å (1 Å=10−10 m) in the cyanobacterium Mastigocladus laminosus and the green alga Chlamydomonas reinhardtii are discussed. The discussion is focused on the binding sites of plastoquinone and quinone analogue inhibitors discerned in the structure. These sites mark the pathway(s) of quinone translocation across the complex.

Binding sites of lipophilic quinone and quinone analogue inhibitors in the cytochrome b6f complex of oxygenic photosynthesis

The main structural features of the cytochrome b6f complex, solved to 3.0-3.1 A (1 A = 10(-10) m) in the cyanobacterium Mastigocladus laminosus and the green alga Chlamydomonas reinhardtii are discussed. The discussion is focused on the binding sites of plastoquinone and quinone analogue inhibitors discerned in the structure. These sites mark the pathway(s) of quinone translocation across the complex.

Evolution of photosynthesis: time-independent structure of the cytochrome b6f complex.

Structures of the cytochrome b(6)f complex obtained from the thermophilic cyanobacterium Mastigocladus laminosus and the green alga Chlamydomonas reinhardtii, whose appearance in evolution is separated by 10(9) years, are almost identical. Two monomers with a molecular weight of 110,000, containing eight subunits and seven natural prosthetic groups, are separated by a large lipid-containing "quinone exchange cavity". A unique heme, heme x, that is five-coordinated and high-spin, with no strong field ligand, occupies a position close to intramembrane heme b(n). This position is filled by the n-side bound quinone, Q(n), in the cytochrome bc(1) complex of the mitochondrial respiratory chain. The structure and position of heme x suggest that it could function in ferredoxin-dependent cyclic electron transport as well as being an intermediate in a quinone cycle mechanism for electron and proton transfer. The significant differences between the cyanobacterial and algal structures are as follows. (i) On the n-side, a plastoquinone molecule is present in the quinone exchange cavity in the cyanobacterial complex, and a sulfolipid is bound in the algal complex at a position corresponding to a synthetic DOPC lipid molecule in the cyanobacterial complex. (ii) On the p-side, in both complexes a quinone analogue inhibitor, TDS, passes through a portal that separates the large cavity from a niche containing the Fe(2)S(2) cluster. However, in the cyanobacterial complex, TDS is in an orientation that is the opposite of its position in the algal structure and bc(1) complexes, so its headgroup in the M. laminosus structure is 20 A from the Fe(2)S(2) cluster.

Efficient large scale purification of his-tagged proton translocating NADH:ubiquinone oxidoreductase (complex I) from the strictly aerobic yeast Yarrowia lipolytica

Proton translocating NADH:ubiquinone oxidoreductase (complex I) is the largest membrane bound multiprotein complex of the respiratory chain and the only one for which no molecular structure is available so far. Thus, information on the mechanism of this central enzyme of aerobic energy metabolism is still very limited. As a new approach to analyze complex I, we have recently established the strictly aerobic yeast Yarrowia lipolytica as a model system that offers a complete set of convenient genetic tools and contains a complex I that is stable after isolation. For crystallization of complex I and to obtain its molecular structure it is a prerequisite to prepare large amounts of highly pure enzyme. Here we present the construction of his-tagged complex I that for the first time allows efficient affinity purification. Our protocol recovers almost 40% of complex I present in Yarrowia mitochondrial membranes. Overall, 40–80 mg highly pure and homogeneous complex I can be obtained from 10 l of an overnight Y. lipolytica culture. After reconstitution into asolectin proteoliposomes, the purified enzyme exhibits full NADH:ubiquinone oxidoreductase activity, is fully sensitive to inhibition by quinone analogue inhibitors and capable of generating a proton-motive force.

Sulfide oxidation in the phototrophic sulfur bacterium Chromatium vinosum.

Sulfide oxidation in the phototrophic purple sulfur bacterium Chromatium vinosum D (DSMZ 180(T)) was studied by insertional inactivation of the fccAB genes, which encode flavocytochrome c, a protein that exhibits sulfide dehydrogenase activity in vitro. Flavocytochrome c is located in the periplasmic space as shown by a PhoA fusion to the signal peptide of the hemoprotein subunit. The genotype of the flavocytochrome-c-deficient Chr. vinosum strain FD1 was verified by Southern hybridization and PCR, and the absence of flavocytochrome c in the mutant was proven at the protein level. The oxidation of thiosulfate and intracellular sulfur by the flavocytochrome-c-deficient mutant was comparable to that of the wild-type. Disruption of the fccAB genes did not have any significant effect on the sulfide-oxidizing ability of the cells, showing that flavocytochrome c is not essential for oxidation of sulfide to intracellular sulfur and indicating the presence of a distinct sulfide-oxidizing system. In accordance with these results, Chr. vinosum extracts catalyzed electron transfer from sulfide to externally added duroquinone, indicating the presence of the enzyme sulfide:quinone oxidoreductase (EC 1.8.5.-). Further investigations showed that the sulfide:quinone oxidoreductase activity was sensitive to heat and to quinone analogue inhibitors. The enzyme is strictly membrane-bound and is constitutively expressed. The presence of sulfide:quinone oxidoreductase points to a connection of sulfide oxidation to the membrane electron transport system at the level of the quinone pool in Chr. vinosum.

Electron spin echo envelope modulation spectroscopy supports the suggested coordination of two histidine ligands to the Rieske iron-sulfur centers of the cytochrome b6f complex on spinach and the cytochrome bc1 complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria

Electron spin echo envelope modulation (ESEEM) experiments performed on the Rieske Fe-S clusters of the cytochrome b6f complex of spinach chloroplasts and of the cytochrome bc1 complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria show modulation components resulting from two distinct classes of 14N ligands. At the g = 1.92 region of the Rieske EPR spectrum of the cytochrome b6f complex, the measured hyperfine couplings for the two classes of coupled nitrogens are A1 = 4.6 MHz and A2 = 3.8 MHz. Similar couplings are observed for the Rieske centers in the three cytochrome bc1 complexes. These ESEEM results indicate a nitrogen coordination environment for these Rieske Fe-S centers that is similar to that of the Fe-S cluster of a bacterial dioxygenase enzyme with two coordinated histidine ligands [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871]. The Rieske Fe-S cluster lacks modulation components from a weakly coupled peptide nitrogen observed in water-soluble spinach ferredoxin. Treatment with the quinone analogue inhibitor DBMIB causes a shift in the Rieske EPR spectrum to g = 1.95 with no alteration in the magnetic coupling to the two nitrogen atoms. However, the ESEEM pattern of the DBMIB-altered Rieske EPR signal shows evidence of an additional weakly coupled nitrogen similar to that observed in the spinach ferredoxin ESEEM patterns.

Studies on the NADH-menaquinone oxidoreductase segment of the respiratory chain in Thermus thermophilus HB-8.

Five distinct low potential iron-sulfur clusters have been identified potentiometrically in the membrane particles from Thermus thermophilus HB-8. Three of these clusters (designated as [N-1H]T, [N-2H]T, and [N-3]T) exhibit the following midpoint redox potentials and g values (Em8.0 = -274 mV, gx,y,z = 1.93, 1.94, 2.02), (Em8.0 = -304 mV, gx,y,z = 1.89, 1.95, 2.04), and (Em8.0 = -289 mV, gx,y,z = 1.80, 1.83, 2.06), respectively. These clusters, one binuclear and two tetranuclear, have been shown to be components of the energy coupled NADH-menaquinone oxidoreductase complex (NADH dh I). They are reducible by NADH in the piericidin A-inhibited aerobic membrane particles as well as in the purified NADH dh I complex. Two additional very low potential iron-sulfur clusters (one binuclear, [N-1L]T, and one tetranuclear, [N-2L]T) were observed in membrane particles. These clusters possess the following physiochemical properties (Em8.0 = -418 mV, gx,y,z = 1.93, 19.5, 2.02) and (Em8.0 = -437 mV, gx,y,z = 1.89, 1.95, 2.04), respectively. No high potential tetranuclear cluster equivalent to the mitochondrial iron-sulfur cluster [N-2]B was found in this bacterial system. In membrane particles isolated from T. thermophilus HB-8 cells, four different semiquinone species have been identified based on their redox midpoint potentials [Em9(Q/QH2) = 40, -100, -160, -300 mV] and sensitivity to the quinone analogue inhibitor, 2-heptyl-4-hydroxy quinoline-N-oxide. Of these semiquinone species the -100 mV component has been suggested to be part of the NADH dehydrogenase. Piericidin A sensitive delta psi formation has been demonstrated to be coupled to the NADH-MQ1 oxidoreductase in membrane vesicles of T. thermophilus HB-8.