Characterization of mutations in crucial residues around the Q(o) binding site of the cytochrome bc complex from Paracoccus denitrificans

The protonation state of residues around the Q(o) binding site of the cytochrome bc(1) complex from Paracoccus denitrificans and their interaction with bound quinone(s) was studied by a combined electrochemical and FTIR difference spectroscopic approach. Site-directed mutations of two groups of conserved residues were investigated: (a) acidic side chains located close to the surface and thought to participate in a water chain leading up to the heme b(L) edge, and (b) residues located in the vicinity of this site. Interestingly, most of the mutants retain a high degree of catalytic activity. E295Q, E81Q and Y297F showed reduced stigmatellin affinity. On the basis of electrochemically induced FTIR difference spectra, we suggest that E295 and D278 are protonated in the oxidized form or that their mutation perturbs protonated residues. Mutations Y302, Y297, E81 and E295, directly perturb signals from the oxidized quinone and of the protein backbone. By monitoring the interaction with the inhibitor stigmatellin for the wild-type enzyme at various redox states, interactions of the bound stigmatellin with amino acid side chains such as protonated acidic residues and the backbone were observed, as well as difference signals arising from the redox active inhibitor itself and the replaced quinone. The infrared difference spectra of the above Q(o) site mutations in the presence of stigmatellin confirm the previously established role of E295 as a direct interaction partner in the enzyme from P.denitrificans as well. The protonated residue E295 is proposed to change the hydrogen-bonding environment upon stigmatellin binding in the oxidized form, and is deprotonated in the reduced form. Of the residues located close to the surface, D278 remains protonated and unperturbed in the oxidized form but its frequency shifts in the reduced form. The mechanistic implications of our observations are discussed, together with previous inhibitor binding data, and referred to the published X-ray structures.     

Determination of the binding rate constants of stigmatellin and UHDBT to bovine cytochrome bc(1) complex by cytochrome c(1) oxidation.

Based on the high electron transfer rate between the [2Fe-2S] cluster and heme c(1) and the elevation of the redox midpoint potential of iron sulfur protein (ISP) upon binding of certain Qo inhibitors, the binding rate constants of stigmatellin and UHDBT to the cytochrome bc(1) complex were determined using a stopped-flow rapid scanning technique. Assuming that the intramolecular electron transfer from ISP to cytochrome c(1) is much faster than the binding of inhibitors, the rate of the inhibitor binding can be determined by the rate of cytochrome c(1) oxidation. The binding rate constants were calculated to be 1.0x10(5) and 2.3x10(5) M(-1) s(-1) at pH 7.5 for stigmatellin and UHDBT, respectively. The binding rate constant of UHDBT is pH dependent and that of stigmatellin is not.     

Modification of the phylloquinone in the A1 binding site in photosystem I studied using time-resolved FTIR difference spectroscopy and density functional theory

A phylloquinone molecule (2-methyl-3-phytyl-1,4-naphthoquinone) occupies the A1 binding site in photosystem I. Previously, we have obtained A1(-)/A1 FTIR difference spectra using labeled and unlabeled photosystem I particles and proposed assignments for many of the bands in the spectra [Sivakumar, V., Wang, R., and Hastings, G. (2005) Biochemistry 44, 1880-1893]. In particular, we suggested that a negative/positive band at 1654/1495 cm(-1) in A1(-)/A1 FTIR DS is due to a C=O/C-:O mode of the neutral/anionic phylloquinone, respectively. To test this hypothesis, we have obtained A1(-)/A1 FTIR DS for menG mutant PS I particles. In menG mutant PS I, phylloquinone in the A1 binding site is replaced with an analogue in which the methyl group at position 2 of the quinone ring is replaced with a hydrogen atom (2-phytyl-1,4-naphthoquinone). In A1(-)/A1 FTIR DS obtained using menG mutant PS I particles, we find that the 1654/1495 cm(-1) bands are upshifted by approximately 6 cm(-1). To test if such upshifts are likely for C=O/C-:O modes of neutral/anionic phylloquinone, we have used density functional theory to calculate the "anion minus neutral" infrared difference spectra for both phylloquinone and its methyl-less analogue. We have also undertaken calculations in which the C4=O carbonyl group of phylloquinone and its methyl-less analogue are hydrogen bonded (to a water or leucine molecule). We find that, irrespective of the hydrogen bonding state of the C4=O group, the C=O/C-:O modes of neutral/reduced phylloquinone are indeed expected to be upshifted by at least 6 cm(-1) upon replacement of the methyl group at position 2 with hydrogen. The calculations also suggest that certain C=C/C-:C modes of neutral/reduced phylloquinone do not shift upon replacement of the methyl group. On the basis of these calculated results, we suggest which bands in the A1(-)/A1 FTIR DS may be associated with C=C/C-:C modes of neutral/reduced phylloquinone, respectively.     

Vibrational modes of ubiquinone in cytochrome bo(3) from Escherichia coli identified by Fourier transform infrared difference spectroscopy and specific (13)C labeling.

In this study we present the infrared spectroscopic characterization of the bound ubiquinone in cytochrome bo(3) from Escherichia coli. Electrochemically induced Fourier transform infrared (FTIR) difference spectra of DeltaUbiA (an oxidase devoid of bound ubiquinone) and DeltaUbiA reconstituted with ubiquinone 2 and with isotopically labeled ubiquinone 2, where (13)C was introduced either at the 1- or at the 4-position of the ring (C=O groups), have been obtained. The vibrational modes of the quinone bound to the discussed high-affinity binding site (Q(H)) are compared to those from the synthetic quinones in solution, leading to the assignment of the C=O modes to a split signal at 1658/1668 cm(-)(1), with both carbonyls similarly contributing. The FTIR spectra of DeltaUbiA reconstituted with the labeled quinones indicate an essentially symmetrical and weak hydrogen bonding of the two C=O groups from the neutral quinone with the protein and distinct conformations of the 2- and 3-methoxy groups. Perturbations of the vibrational modes of the 5-methyl side groups are discussed for a signal at 1452 cm(-)(1). Only negligible shifts of the aromatic ring modes can be reported for the reduced and the protonated form of the quinone. Alterations of the protein upon quinone binding are reflected in the electrochemically induced FTIR difference spectra. In particular, difference signals at 1640-1633 cm(-)(1) and 1700-1670 cm(-)(1) indicate variations of beta-sheet secondary structure elements and loops, bands at 1706 and 1678 cm(-)(1) are tentatively attributed to individual amino acids, and a difference signal a 1540 cm(-)(1) is discussed to reflect an influence on C=C modes of the porphyrin ring or on deprotonated propionate groups of the hemes. Further tentative assignments are presented and discussed. The (13)C labeling experiments allow the assignment of the vibrational modes of a bound ubiquinone 8 in the electrochemically induced FTIR difference spectra of wild-type bo(3).     

Regulatory interactions between ubiquinol oxidation and ubiquinone reduction sites in the dimeric cytochrome bc1 complex

We have obtained evidence for conformational communication between ubiquinol oxidation (center P) and ubiquinone reduction (center N) sites of the yeast bc1 complex dimer by analyzing antimycin binding and heme bH reduction at center N in the presence of different center P inhibitors. When stigmatellin was occupying center P, concentration-dependent binding of antimycin occurred only to half of the center N sites. The remaining half of the bc1 complex bound antimycin with a slower rate that was independent of inhibitor concentration, indicating that a slow conformational change needed to occur before half of the enzyme could bind antimycin. In contrast, under conditions where the Rieske protein was not fixed proximal to heme bL at center P, all center N sites bound antimycin with fast and concentration-dependent kinetics. Additionally, the extent of fast cytochrome b reduction by menaquinol through center N in the presence of stigmatellin was approximately half of that observed when myxothiazol was bound at center P. The reduction kinetics of the bH heme by decylubiquinol in the presence of stigmatellin or myxothiazol were also consistent with a model in which fixation of the Rieske protein close to heme bL in both monomers allows rapid binding of ligands only to one center N. Decylubiquinol at high concentrations was able to abolish the biphasic binding of antimycin in the presence of stigmatellin but did not slow down antimycin binding rates. These results are discussed in terms of half-of-the-sites activity of the dimeric bc1 complex.     

Conformational changes in the cytochrome b6f complex induced by inhibitor binding

Binding of stigmatellin, an inhibitor of the Q(o) site of the bc-type complexes, has been shown to induce large conformational changes of the Rieske protein in the respiratory bc(1) complex (Kim, H., Xia, D., Yu, C. A., Xia, J. Z., Kachurin, A. M., Zhang, L., Yu, L., and Deisenhofer, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8026-8033; Iwata, S., Lee, J. W., Okada, K., Lee, J. K., Iwata, M., Rasmussen, B., Link, T. A., Ramaswamy, S., and Jap, B. K. (1998) Science 281, 64-71; Zhang, Z., Huang, L., Shulmeister, V. M., Chi, Y. I., Kim, K. K., Hung, L. W., Crofts, A. R., Berry, E. A., and Kim, S. H. (1998) Nature 392, 677-684). Such a movement seems necessary to shuttle electrons from the membrane-soluble quinol to the extramembrane heme of cytochrome c(1). To see whether similar changes occur in the related photosynthetic b(6)f complex, we have studied the effect of the binding of stigmatellin to the eukaryotic b(6)f complex by electron crystallography. Comparison of projection maps of thin three-dimensional crystals prepared with or without stigmatellin, and either negatively stained or embedded in glucose, reveals a similar type of movement to that observed in the bc(1) complex and suggests also the occurrence of conformational changes in the transmembrane region.     

FTIR difference spectroscopy in combination with isotope labeling for identification of the carbonyl modes of P700 and P700+ in photosystem I

Room temperature, light induced (P700(+)-P700) Fourier transform infrared (FTIR) difference spectra have been obtained using photosystem I (PS I) particles from Synechocystis sp. PCC 6803 that are unlabeled, uniformly (2)H labeled, and uniformly (15)N labeled. Spectra were also obtained for PS I particles that had been extensively washed and incubated in D(2)O. Previously, we have found that extensive washing and incubation of PS I samples in D(2)O does not alter the (P700(+)-P700) FTIR difference spectrum, even with approximately 50% proton exchange. This indicates that the P700 binding site is inaccessible to solvent water. Upon uniform (2)H labeling of PS I, however, the (P700(+)-P700) FTIR difference spectra are considerably altered. From spectra obtained using PS I particles grown in D(2)O and H(2)O, a ((1)H-(2)H) isotope edited double difference spectrum was constructed, and it is shown that all difference bands associated with ester/keto carbonyl modes of the chlorophylls of P700 and P700(+) downshift 4-5/1-3 cm(-1) upon (2)H labeling, respectively. It is also shown that the ester and keto carbonyl modes of the chlorophylls of P700 need not be heterogeneously distributed in frequency. Finally, we find no evidence for the presence of a cysteine mode in our difference spectra. The spectrum obtained using (2)H labeled PS I particles indicates that a negative difference band at 1698 cm(-1) is associated with at least two species. The observed (15)N and (2)H induced band shifts strongly support the idea that the two species are the 13(1) keto carbonyl modes of both chlorophylls of P700. We also show that a negative difference band at approximately 1639 cm(-1) is somewhat modified in intensity, but unaltered in frequency, upon (2)H labeling. This indicates that this band is not associated with a strongly hydrogen bonded keto carbonyl mode of one of the chlorophylls of P700.     

Asymmetric binding of stigmatellin to the dimeric Paracoccus denitrificans bc1 complex: evidence for anti-cooperative ubiquinol oxidation and communication between center P ubiquinol oxidation sites

We have investigated the mechanism responsible for half-of-the-sites activity in the dimeric cytochrome bc(1) complex from Paracoccus denitrificans by characterizing the kinetics of inhibitor binding to the ubiquinol oxidation site at center P. Both myxothiazol and stigmatellin induced a 2-3 nm shift of the visible absorbance spectrum of the b(L) heme. The shift generated by myxothiazol was symmetric, with monophasic kinetics that indicate equal binding of this inhibitor to both center P sites. In contrast, stigmatellin generated an asymmetric shift in the b(L) spectrum, with biphasic kinetics in which each phase contributed approximately half of the total magnitude of the spectral change. The faster binding phase corresponded to a more symmetrical shift of the b(L) spectrum relative to the slower binding phase, indicating that approximately half of the center P sites bound stigmatellin more slowly and in a different position relative to the b(L) heme, generating a different effect on its electronic environment. Significantly, the slow stigmatellin binding phase was lost as the inhibitor concentration was increased. This implies that a conformational change is transmitted from one center P site in the dimer to the other upon stigmatellin binding to one monomer, rendering the second site less accessible to the inhibitor. Because the position that stigmatellin occupies at center P is considered to be analogous to that of the quinol substrate at the moment of electron transfer, these results indicate that the productive enzyme-substrate configuration is prevented from occurring in both monomers simultaneously.     

FTIR spectroscopic evidence for the involvement of an acidic residue in quinone binding in cytochrome bd from Escherichia coli

In this work, FTIR difference spectroscopy is used to search for possible binding partners and protonable groups involved in the binding of the quinol to cytochrome bd from Escherichia coli. In addition, the electrochemically induced FTIR difference spectra are compared for preparations of the enzyme isolated from cells grown at different oxygen levels in which the quinone content of the membrane is altered. On this basis, difference signals can be tentatively attributed to the vibrational modes of the different quinones types that are associated with the enzyme depending on growth conditions. Furthermore, vibrational modes due to the redox-dependent reorganization of the protein vary depending on the quinone associated with the isolated enzyme. Of particular interest are the observations that a mode at 1738 cm(-1) is decreased and a mode at 1595 cm(-1) is increased as observed in direct comparison to the data obtained from samples grown anaerobically. These signals indicate a change in the protonation state of an aspartic or glutamic acid. Since these changes are observed when the ubiquinone ratio in the preparation increases, the data provide evidence for the modulation of the binding site by the interacting quinone and the involvement of an acidic group in the binding site. The tentative assignments of the vibrational modes are supported by electrochemically induced FTIR difference spectra of cytochrome bd in the presence of the specific quinone binding site inhibitors heptylhydroxyquinoline-N-oxide (HQNO) or 2-methyl-3-undecylquinolone-4. Whereas HQNO leads to strong shifts in the FTIR redox difference spectrum, 2-methyl-3-undecylquinolone-4 induces a specific shift of a mode at 1635 cm(-1), which likely originates from the displacement of the C=O group of the bound quinone.     

Resonance Raman and Fourier transform infrared spectroscopic studies of the acyl carbonyl group in [3-(5-methyl-2-thienyl)acryloyl]chymotrypsin: evidence for artifacts in the spectra obtained by both techniques

The acyl carbonyl group of [3-(5-methyl-2-thienyl)acryloyl]chymotrypsin (5MeTA-chymotrypsin) has been investigated by using both resonance Raman (RR) and Fourier transform infrared (FTIR) spectroscopies. The spectrum of the acyl-enzyme carbonyl group has been obtained as a function of pH over the range 3.0-10.0 in the RR experiments and over the range 3.4-7.6 (p2H) in the FTIR experiments. The carbonyl spectral profiles obtained by using FTIR spectroscopy are substantially different from the carbonyl profiles obtained by using RR spectroscopy. The FTIR spectra were obtained by subtracting the spectrum of the free enzyme from that of the acyl-enzyme. Use of the active-site inhibitor phenylmethanesulfonyl fluoride demonstrates that part of the intensity observed in the FTIR spectra of 5MeTA-chymotrypsin is due to a subtraction artifact giving rise to enzyme-associated bands, probably from peptide groups perturbed by substrate binding. The enzyme bands can be removed by subtracting the FTIR spectrum of 13C=O acyl-enzyme from that of 12C=O acyl-enzyme. Additionally, this procedure reveals that one of the acyl-enzyme carbonyl bands observed at 1727 cm-1 using RR spectroscopy is absent in the FTIR acyl-enzyme spectrum. However, a feature near 1720 cm-1 can be induced in the FTIR spectrum by actinic light in the near-UV region. Thus, it is proposed that the 1727 cm-1 RR carbonyl band results from a population of acyl-enzymes which is generated by exposure to the laser beam during RR data collection. When both the RR and FTIR data are adjusted to remove artifacts, they provide essentially identical carbonyl stretching profiles.     

The interaction of the Rieske iron-sulfur protein with occupants of the Qo-site of the bc1 complex, probed by electron spin echo envelope modulation.

The bifurcated reaction at the Q(o)-site of the bc(1) complex provides the mechanistic basis of the proton pumping activity through which the complex conserves redox energy in the proton gradient. Structural information about the binding of quinone at the site is lacking, because the site is vacant in crystals of the native complexes. We now report the first structural characterization of the interaction of the native quinone occupant with the Rieske iron-sulfur protein in the bc(1) complex of Rhodobacter sphaeroides, using high resolution EPR. We have compared the binding configuration in the presence of quinone with the known structures for the complex with stigmatellin and myxothiazol. We have shown by using EPR and orientation-selective electron spin echo envelope modulation (ESEEM) measurements of the iron-sulfur protein that when quinone is present in the site, the isotropic hyperfine constant of one of the N(delta) atoms of a liganding histidine of the [2Fe-2S] cluster is similar to that observed when stigmatellin is present and different from the configuration in the presence of myxothiazol. The spectra also show complementary differences in nitrogen quadrupole splittings in some orientations. We suggest that the EPR characteristics, the ESEEM spectra, and the hyperfine couplings reflect a similar interaction between the iron-sulfur protein and the quinone or stigmatellin and that the N(delta) involved is that of a histidine (equivalent to His-161 in the chicken mitochondrial complex) that forms both a ligand to the cluster and a hydrogen bond with a carbonyl oxygen atom of the Q(o)-site occupant.     

Functional coupling of PSST and ND1 subunits in NADH:ubiquinone oxidoreductase established by photoaffinity labeling.

NADH:ubiquinone oxidoreductase (complex I) is the first, largest and most complicated enzyme of the mitochondrial electron transport chain. Photoaffinity labeling with the highly potent and specific inhibitor trifluoromethyldiazirinyl-[(3)H]pyridaben ([(3)H]TDP) labels only the PSST and ND1 subunits of complex I in electron transport particles. PSST is labeled at a high-affinity site responsible for inhibition of enzymatic activity while ND1 is labeled at a low-affinity site not related to enzyme inhibition. In this study we found, as expected, that 13 complex I inhibitors decreased labeling at the PSST site without effect on ND1 labeling. However, there were striking exceptions where an apparent interaction was found between the PSST and ND1 subunits: preincubation with NADH increases PSST labeling and decreases ND1 labeling; the very weak complex I inhibitor 1-methyl-4-phenylpyridinium ion (MPP(+)) and the semiquinone analogue stigmatellin show the opposite effect with increased labeling at ND1 coupled to decreased labeling at PSST in a concentration- and time-dependent manner. MPP(+), stigmatellin and ubisemiquinone have similarly positioned centers of highly negative and positive electrostatic potential surfaces. Perhaps the common action of MPP(+) and stigmatellin on the functional coupling of the PSST and ND1 subunits is initiated by binding at a semiquinone binding site in complex I.     

Stigmatellin induces reduction of iron-sulfur protein in the oxidized cytochrome bc1 complex

Stigmatellin, a Q(P) site inhibitor, inhibits electron transfer from iron-sulfur protein (ISP) to cytochrome c1 in the bc1 complex. Stigmatellin raises the midpoint potential of ISP from 290 mV to 540 mV. The binding of stigmatellin to the fully oxidized complex, oxidized completely by catalytic amounts of cytochrome c oxidase and cytochrome c, results in ISP reduction. The extent of ISP reduction is proportional to the amount of inhibitor used and reaches a maximum when the ratio of inhibitor to enzyme complex reaches unity. A g = 2.005 EPR peak, characteristic of an organic free radical, is also observed when stigmatellin is added to the oxidized complex, and its signal intensity depends on the amount of stigmatellin. Addition of ferricyanide, a strong oxidant, to the oxidized complex also generates a g = 2.005 EPR peak that is oxidant concentration-dependent. Oxygen radicals are generated when stigmatellin is added to the oxidized complex in the absence of the exogenous substrate, ubiquinol. The amount of oxygen radical formed is proportional to the amount of stigmatellin added. Oxygen radicals are not generated when stigmatellin is added to a mutant bc1 complex lacking the Rieske iron-sulfur cluster. Based on these results, it is proposed that ISP becomes a strong oxidant upon stigmatellin binding, extracting electrons from an organic compound, likely an amino acid residue. This results in the reduction of ISP and generation of organic radicals.     

Natural resistance to inhibitors of the ubiquinol cytochrome c oxidoreductase of Rubrivivax gelatinosus: sequence and functional analysis of the cytochrome bc(1) complex

Biochemical analyses of Rubrivivax gelatinosus membranes have revealed that the cytochrome bc(1) complex is highly resistant to classical inhibitors including myxothiazol, stigmatellin, and antimycin. This is the first report of a strain exhibiting resistance to inhibitors of both catalytic Q(0) and Q(i) sites. Because the resistance to cytochrome bc(1) inhibitors is primarily related to the cytochrome b primary structure, the petABC operon encoding the subunits of the cytochrome bc(1) complex of Rubrivivax gelatinosus was sequenced. In addition to homologies to the corresponding proteins from other organisms, the deduced amino acid sequence of the cytochrome b polypeptide shows (i) an E303V substitution in the highly conserved PEWY loop involved in quinol/stigmatellin binding, (ii) other substitutions that could be involved in resistance to cytochrome bc(1) inhibitors, and (iii) 14 residues instead of 13 between the histidines in helix IV that likely serve as the second axial ligand to the b(H) and b(L) hemes, respectively. These characteristics imply different functional properties of the cytochrome bc(1) complex of this bacterium. The consequences of these structural features for the resistance to inhibitors and for the properties of R. gelatinosus cytochrome bc(1) are discussed with reference to the structure and function of the cytochrome bc(1) complexes from other organisms.     

The inhibitor DBMIB provides insight into the functional architecture of the Qo site in the cytochrome b6f complex

Previously [Roberts, A. G., and Kramer, D. M. (2001) Biochemistry 40, 13407-13412], we showed that 2 equiv of the quinone analogue 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) could occupy the Q(o) site of the cytochrome (cyt) b(6)f complex simultaneously. In this work, a study of electron paramagnetic resonance (EPR) spectra from the oriented cyt b(6)f complex shows that the Rieske iron-sulfur protein (ISP) is in distinct orientations, depending on the stoichiometry of the inhibitor at the Q(o) site. With a single DBMIB at the Q(o) site, the ISP is oriented with the 2Fe-2S cluster toward cyt f, which is similar to the orientation of the ISP in the X-ray crystal structure of the cyt b(6)f complex from thermophilic cyanobacterium Mastigocladus laminosus in the presence of DBMIB, as well as that of the chicken mitochondrial cyt bc(1) complex in the presence of the class II inhibitor myxothiazol, which binds in the so-called "proximal niche", near the cyt b(L) heme. These data suggest that the high-affinity DBMIB site is at the proximal niche Q(o) pocket. With >or=2 equiv of DBMIB bound, the Rieske ISP is in a position that resembles the ISP(B) position of the chicken mitochondrial cyt bc(1) complex in the presence of stigmatellin and the Chlamydomonas reinhardtii cyt b(6)f complex in the presence of tridecylstigmatellin (TDS), which suggests that the low-affinity DBMIB site is at the distal niche. The close interaction of DBMIB bound at the distal niche with the ISP induced the well-known effects on the 2Fe-2S EPR spectrum and redox potential. To further test the effects of DBMIB on the ISP, the extents of cyt f oxidation after flash excitation in the presence of photosystem II inhibitor DCMU were measured as a function of DBMIB concentration in thylakoids. Addition of DBMIB concentrations at which a single binding was expected did not markedly affect the extent of cyt f oxidation, whereas higher concentrations, at which double occupancy was expected, increased the extent of cyt f oxidation to levels similar to that of cyt f oxidation in the presence of a saturating concentration of stigmatellin. Simulations of the EPR g-tensor orientations of the 2Fe-2S cluster versus the physical orientations based on single-crystal studies of the cyt bc(1) complex suggest that the soluble ISP domain of the spinach cyt b(6)f complex can rotate by at least 53 degrees, which is consistent with long-range ISP domain movement. Implications of these results are discussed in the context of the X-ray crystal structures of the chicken mitochondrial cyt bc(1) complex and the M. laminosus and C. reinhardtii cyt b(6)f complexes.     

Plasmon waveguide resonance spectroscopic evidence for differential binding of oxidized and reduced Rhodobacter capsulatus cytochrome c2 to the cytochrome bc1 complex mediated by the conformation of the Rieske iron-sulfur protein

The dissociation constants for the binding of Rhodobacter capsulatus cytochrome c2 and its K93P mutant to the cytochrome bc1 complex embedded in a phospholipid bilayer were measured by plasmon waveguide resonance spectroscopy in the presence and absence of the inhibitor stigmatellin. The reduced form of cytochrome c2 strongly binds to reduced cytochrome bc1 (Kd = 0.02 microM) but binds much more weakly to the oxidized form (Kd = 3.1 microM). In contrast, oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and 0.58 microM. Such a biphasic interaction is consistent with binding to two separate sites or conformations of oxidized cytochrome c2 and/or cytochrome bc1. However, in the presence of stigmatellin, we find that oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a monophasic fashion with high affinity (Kd = 0.06 microM) and reduced cytochrome c2 binds less strongly (Kd = 0.11 microM) but approximately 30-fold more tightly than in the absence of stigmatellin. Structural studies with cytochrome bc1, with and without the inhibitor stigmatellin, have led to the proposal that the Rieske protein is mobile, moving between the cytochrome b and cytochrome c1 components during turnover. In one conformation, the Rieske protein binds near the heme of cytochrome c1, while the cytochrome c2 binding site is also near the cytochrome c1 heme but on the opposite side from the Rieske site, where cytochrome c2 cannot directly interact with Rieske. However, the inhibitor, stigmatellin, freezes the Rieske protein iron-sulfur cluster in a conformation proximal to cytochrome b and distal to cytochrome c1. We conclude from this that the dual conformation of the Rieske protein is primarily responsible for biphasic binding of oxidized cytochrome c2 to cytochrome c1. This optimizes turnover by maximizing binding of the substrate, oxidized cytochrome c2, when the iron-sulfur cluster is proximal to cytochrome b and minimizing binding of the product, reduced cytochrome c2, when it is proximal to cytochrome c1.     

A comparison of stigmatellin conformations, free and bound to the photosynthetic reaction center and the cytochrome bc1 complex

We describe in detail the conformations of the inhibitor stigmatellin in its free form and bound to the ubiquinone-reducing (Q(B)) site of the reaction center and to the ubiquinol-oxidizing (Q(o)) site of the cytochrome bc(1) complex. We present here the first structures of a stereochemically correct stigmatellin in complexes with a bacterial reaction center and the yeast cytochrome bc1 complex. The conformations of the inhibitor bound to the two enzymes are not the same. We focus on the orientations of the stigmatellin side-chain relative to the chromone head group, and on the interaction of the stigmatellin side-chain with these membrane protein complexes. The different conformations of stigmatellin found illustrate the structural variability of the Q sites, which are affected by the same inhibitor. The free rotation about the chi1 dihedral angle is an essential factor for allowing stigmatellin to bind in both the reaction center and the cytochrome bc1 pocket.     

Primary donor photo-oxidation in photosystem I: a re-evaluation of (P700(+) - P700) Fourier transform infrared difference spectra.

Light-induced Fourier transform infrared (FTIR) difference spectroscopy has been used to study the photo-oxidation of the primary electron donor (P700) in PS I particles from Chlamydomonas reinhardtii and Synechocystis sp. PCC 6803. To aid in the interpretation of the spectra, PS I particles from a site-directed mutant of C. reinhardtii, in which the axial histidine ligand (HisA676) was changed to serine, were also studied. A high-frequency (3300-2600 cm(-1)) electronic transition is observed for all PS I particles, demonstrating that P700 is dimeric. The electronic band is, however, species-dependent, indicating some differences in the electronic structure of P700 and/or P700(+) in C. reinhardtii and Synechocystis sp. 6803. For PS I particles from C. reinhardtii, substitution of HisA676 with serine has little effect on the ester carbonyl modes of the chlorophylls of P700. However, the keto carbonyl modes are considerably altered. Comparison of (P700(+) - P700) FTIR difference spectra obtained using PS I particles from the wild type (WT) and the HS(A676) mutant of C. reinhardtii indicates that the mutation primarily exerts its influence on the P700 ground state. The 13(1) keto carbonyls of the chlorophylls of P700 of the wild type absorb at similar frequencies, which has previously made these transitions difficult to resolve. However, for the HS(A676) mutant, the 13(1) keto carbonyl of chlorophyll a or chlorophyll a' of P700 on PsaB or PsaA absorbs at 1703.4 or 1694.2 cm(-1), respectively, allowing their unambiguous resolution. Upon P700(+) formation, in both PS I particles from C. reinhardtii, the higher-frequency carbonyl band upshifts by approximately 14 cm(-1) while the lower frequency carbonyl downshifts by approximately 10 cm(-1). The similarity in the spectra for WT PS I particles from C. reinhardtii and Synechocystis sp. 6803 indicates that a similar interpretation is probably valid for PS I particles from both species. The mutant results allow for an interpretation of the behavior of the 13(1) keto carbonyls of P700 that is different from previous work [Breton, J., Nabedryk, E., and Leibl, W. (1999) Biochemistry 38, 11585-11592], in which it was suggested that 13(1) keto carbonyls of P700 absorb at 1697 and 1639 cm(-1), and upshift by 21 cm(-1) upon cation formation. The interpretation of the spectra reported here is more in line with recent results from ENDOR spectroscopy and high-resolution crystallography.     

Certain metal ions are inhibitors of cytochrome b6f complex 'Rieske' iron-sulfur protein domain movements

Many current models of the Q cycle for the cytochrome (cyt) b6f and the cyt bc1 complexes incorporate 'Rieske' iron-sulfur protein (ISP) domain movements to gate electron transfer and to ensure high yields of proton shuttling. It was previously proposed that copper ions, which bind at a site distant from the quinol oxidase (Q(o)) site, inhibit plastoquinol (PQH2) binding by restraining the hydrophilic head domain of the ISP [Rao B. K., S., Tyryshkin, A. M., Roberts, A. G., Bowman, M. K., and Kramer, D. M. (1999) Biochemistry 38, 3285-3296]. The present work presents evidence that this is indeed the case for both copper ions and Zn2+, which appear to inhibit by similar mechanisms. Electron paramagnetic resonance (EPR) spectra show that Cu2+ and Zn2+ binding to the cyt b6f complex displaces the Q(o) site inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB). At high concentrations, both DBMIB and Cu2+ or Zn2+ can bind simultaneously, altering the Rieske 2Fe2S cluster and Cu2+ EPR spectra, suggesting perturbations in their respective binding sites. Both Zn2+ and Cu1+ altered the orientations of the Rieske 2Fe2S cluster with respect to the membrane plane, but had no effect on that of the cyt b6 hemes. Cu2+ was found to change the orientation of the cyt f heme plane, consistent with binding on the cyt f protein. Within conservative constraints, the data suggest that the ISP is shifted into a position intermediate between the ISP(C) position, when the Q(o) site is unoccupied, and the ISP(B) position, when the Q(o) site is occupied by inhibitors such as DBMIB or stigmatellin. These results support the role of ISP domain movements in Q(o) site catalysis.     

Inhibitor effects on redox-linked protonations of the b haems of the mitochondrial bc1 complex

The effects of pH and inhibitors on the spectra and redox properties of the haems b of the bc1 complex of beef heart submitochondrial particles were investigated. The major findings were: (1) both haems have a weakly redox-linked protonatable group with pKox and pKred of around 6 and 8; (2) at pH values above 7, haem bH becomes heterogeneous in its redox behaviour. This heterogeneity is removed by the Qi site inhibitors antimycin A, funiculosin and HQNO, but not by the Qo site inhibitors myxothiazol or stigmatellin; (3) of all inhibitors tested only funiculosin had a large effect on the Em/pH profile of either haem b. In all cases where definite effects were found, the haem most affected was that thought to be closest to the site of inhibitor binding; (4) spectral shifts of haem groups caused by inhibitor binding were usually, but not always, of the haem group closest to the binding site; (5) titrations with succinate/fumarate were in reasonable agreement with redox-mediated data provided that strict anaerobiosis was maintained. Apparent large shifts of haem midpoint potentials with antimycin A and myxothiazol could be produced in aerobic succinate/fumarate titrations in the presence of cyanide, as already reported in the literature, but these were artefactual; (6) the heterogeneous haem bH titration behaviour can be simulated with a model similar to that proposed by Salerno et al. (J. Biol. Chem. (1989) 264, 15398-15403) in which there is redox interaction between haem bH and ubiquinone species bound at the Qi site. Simulations closely fit both the haem bH data and known semiquinone data only if it is assumed that semiquinone bound to oxidised haem bH is EPR-silent.