Direct evidence for the interaction of stigmatellin with a protonated acidic group in the bc(1) complex from Saccharomyces cerevisiae as monitored by FTIR difference spectroscopy and 13C specific labeling

In this study a combined electrochemical and FTIR spectroscopic approach was applied to monitor the binding of stigmatellin, a Q(o) site inhibitor of the cytochrome bc(1) complex from Saccharomyces cerevisiae. Natural stigmatellin A induced clear shifts in the redox-induced FTIR difference spectra. For data interpretation a stigmatellin derivative (UST) with the conjugated trienes replaced by an aliphatic tail was synthesized, and the carbonyl group shown in crystal structures to interact with His181, the [2Fe-2S] ligand of the Rieske, was specifically (13)C labeled. Electrochemically induced FTIR difference spectra of the inhibitors in CH(3)OD were obtained and revealed signals characteristic for the oxidized and reduced forms of the labeled and unlabeled compounds. On the basis of signals from the inhibitors alone, the binding of the inhibitor to the bc(1) complex was monitored. Direct evidence for the interaction of the carbonyl group with the protein was provided by the observed shift of the nu(C=O) vibrational mode of about 10 cm(-1). In addition, redox-dependent reorganizations of the protein were identified, including protonation changes of acidic residues at 1746 and 1734 cm(-1). The conformational changes observed upon inhibitor binding are discussed with respect to the crystal structures and proposed mechanistic models [Hunte, C., Koepke, J., Lange, C., Rossmanith, T., and Michel, H. (2000) Structure 8, 669-684; Palsdottir, H., Lojero, C. G., Trumpower, B. L., and Hunte, C. (2003) J. Biol. Chem. 278, 31303-31311].     

Structural basis for the quinone reduction in the bc1 complex: a comparative analysis of crystal structures of mitochondrial cytochrome bc1 with bound substrate and inhibitors at the Qi site

Cytochrome bc(1) is an integral membrane protein complex essential to cellular respiration and photosynthesis. The Q cycle reaction mechanism of bc(1) postulates a separated quinone reduction (Q(i)) and quinol oxidation (Q(o)) site. In a complete catalytic cycle, a quinone molecule at the Q(i) site receives two electrons from the b(H) heme and two protons from the negative side of the membrane; this process is specifically inhibited by antimycin A and NQNO. The structures of bovine mitochondrial bc(1) in the presence or absence of bound substrate ubiquinone and with either the bound antimycin A(1) or NQNO were determined and refined. A ubiquinone with its first two isoprenoid repeats and an antimycin A(1) were identified in the Q(i) pocket of the substrate and inhibitor bound structures, respectively; the NQNO, on the other hand, was identified in both Q(i) and Q(o) pockets in the inhibitor complex. The two inhibitors occupied different portions of the Q(i) pocket and competed with substrate for binding. In the Q(o) pocket, the NQNO behaves similarly to stigmatellin, inducing an iron-sulfur protein conformational arrest. Extensive binding interactions and conformational adjustments of residues lining the Q(i) pocket provide a structural basis for the high affinity binding of antimycin A and for phenotypes of inhibitor resistance. A two-water-mediated ubiquinone protonation mechanism is proposed involving three Q(i) site residues His(201), Lys(227), and Asp(228).     

Role of the PEWY glutamate in hydroquinone-quinone oxidation-reduction catalysis in the Qo Site of cytochrome bc1

The glutamic acid residue of the conserved PEWY motif of the Q(o) site of cytochrome bc(1) is widely discussed as central to reversible Q(o) site catalysis of two-electron, two-proton hydroquinone-quinone oxidation-reduction. Extensive mutation of this glutamate (E295) to A, V, F, H, K, and Q in purple photosynthetic Rhodobacter capsulatus results in hydroquinone oxidation rates that are between 5 and 50-fold slower than that in the wild type. However, the mutants show little or no detectable effects on hydroquinone or quinone exchange and binding at the Q(o) site nor on subsequent Q(o) site-mediated redox equilibria in the c-chain and b-chain from pH 5-10. Lack of effects of mutations on the E(m)/pH plots rules out involvement of E295 in the strong electron-proton coupling evident in either the FeS center or heme b(L). These detailed equilibrium and kinetic analyses demonstrate that E295 is not irreplaceable in the Q(o) site catalytic mechanism. Rather, E295 and several other Q(o) site residues that can also be widely varied and still support hydroquinone oxidation illustrate the considerable resilience of Q(o) site activity to mutational change in Q(o) site environs. Residues and water molecules appear to cooperate in providing a physical and chemical environment supporting hydroquinone oxidation rates comparable to those seen in nonprotein aqueous environments at electrodes. We suggest that residues at the Q(o) site (and, possibly, other respiratory and photosynthetic quinone and oxygen binding sites) are a product of natural selection primarily acting not to lower catalytic barriers according to the traditional view of enzymatic catalysis but rather to develop specificity by raising barriers in defense of semiquinone loss or energy wasting short-circuit reactions.     

Amino acid residues involved in stereoselective inhibition of cholinesterases with bambuterol

Bambuterol is a chiral carbamate known as selective inhibitor of butyrylcholinesterase (BChE). In order to relate bambuterol selectivity and stereoselectivity of cholinesterases to the active site residues, we studied the inhibition of recombinant mouse BChE, acetylcholinesterase (AChE) and six AChE mutants, employed to mimic BChE active site residues, by bambuterol enantiomers. Both enantiomers selectively inhibited BChE about 8000 times faster than AChE. The largest inhibition rate increase in comparison to AChE w.t. was observed with the F295L/Y337A mutant, showing that leucine 295 and alanine 337 are crucial residues in BChE for high bambuterol selectivity. All studied enzymes preferred inhibition by the R- over the S-bambuterol. The enlargement of the AChE choline binding site and of the acyl pocket by single or double mutations (Y337A, F295L/Y337A and F297I/Y337A) increased, in comparison to w.t. enzymes, inhibition rate constants of R- bambuterol more than that of S- bambuterol resulting in four times higher stereoselectivity. Peripheral site mutations (Y124Q and Y72N/Y124Q/Y337A) increased inhibition rate by S- more than R-bambuterol and consequently diminished the stereoselectivity.     

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.     

Modulation of the midpoint potential of the [2Fe-2S] Rieske iron sulfur center by Qo occupants in the bc1 complex

Following addition of myxothiazol to antimycin-treated chromatophores from Rhodobacter sphaeroides poised at an ambient redox potential (E(h)) of approximately 300 mV, the amplitude of the flash-induced cytochrome c(1) oxidation in the ms range increased, indicating a decrease in the availability of electrons from the immediate donor to c(1), the Rieske iron-sulfur protein (ISP). Because the effect was seen only over the limited E(h) range, we conclude that it is due to a decrease in the apparent midpoint redox potential (E(m)) of the ISP by about 40 mV on addition of myxothiazol. This is in line with the change in E(m) previously seen in direct redox titrations. Our results show that the reduced ISP binds with quinone at the Q(o) site with a higher affinity than does the oxidized ISP. The displacement of ubiquinone by myxothiazol leads to elimination of this preferential binding of the ISP reduced form and results in a shift in the midpoint potential of ISP to a more negative value. A simple hypothesis to explain this effect is that myxothiazol prevents formation of hydrogen bond of ubiquinone with the reduced ISP. We conclude that all Q(o) site occupants (ubiquinone, UHDBT, stigmatellin) that form hydrogen bonds with the reduced ISP shift the apparent E(m) of the ISP in the same direction to more positive values. Inhibitors that bind in the domain of the Q(o) site proximal to heme b(L) (myxothiazol, MOA-stilbene) and displace ubiquinone from the site cause a decrease in E(m) of ISP. We present a new formalism for treatment of the relation between E(m) change and the binding constants involved, which simplifies analysis. Using this formalism, we estimated that binding free energies for hydrogen bond formation with the Q(o) site occupant, range from the largest value of approximately 23 kJ mol(-1) in the presence of stigmatellin (appropriate for the buried hydrogen bond shown by structures), to a value of approximately 3.5 kJ mol(-1) in the native complex. We discuss this range of values in the context of a model in which the native structure constrains the interaction of ISP with the Q(o) site occupant so as to favor dissociation and the faster kinetics of unbinding necessary for rapid turnover.     

Interactions between the donor and acceptor sides in bacterial reaction centers

The apparent equilibrium constant K'(2) for electron transfer between the primary (Q(A)) and secondary (Q(B)) quinone acceptors of the reaction center was measured in chromatophores of Rhodobacter capsulatus. In the presence of the oxidized primary donor P(+), we obtained a value of K'(2)(P(+)) approximately 100 at pH 7.2, based on the rates of recombination from P(+)Q(A-) and P(+)Q(B-). K'(2) was also measured in the presence of reduced P, from the damping of semiquinone oscillations during a series of single turnover flashes. A 5-fold smaller value, K'(2)(P) approximately 20, was found. Additional information on the interactions between the donor and acceptor sides was obtained by measuring the shift of the midpoint potential of P caused by the presence of Q(B-) or Q(A-)S (where S indicates the presence of the inhibitor stigmatellin). A stabilization of the oxidized state P(+) was observed in both instances, by 10 mV for Q(B-) and 30 mV for Q(A-)S. The larger stabilization of P(+)Q(A-)S with respect to P(+)Q(B-) does not account for the effect of P(+)/P on K'(2). Analysis of these results indicates that the interactions between P(+)/P and Q(A)/Q(A)(-) are markedly modified depending on the occupancy of the Q(B) pocket by ubiquinone or by stigmatellin. We propose that the large value of K'(2)(P(+)) results essentially from a conformational destabilization of the P(+)Q(A-) state, that is relieved when the proximal site of the Q(B) pocket is occupied by stigmatellin.     

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.     

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.     

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.     

Tight binding of inhibitors to bovine bc1 complex is independent of the Rieske protein redox state. Consequences for semiquinone stabilization in the quinol oxidation site

To determine the effect of the redox state of the Rieske protein on ligand binding to the quinol oxidation site of the bc(1) complex, we measured the binding rate constants (k(1)) for stigmatellin and myxothiazol, at different concentrations of decylbenzoquinone or decylbenzoquinol, in the bovine bc(1) complex with the Rieske protein in the oxidized or reduced state. Stigmatellin and myxothiazol bound tightly and competitively with respect to quinone or quinol, independently of the redox state of the Rieske protein. In the oxidized bc(1) complex, the k(1) values for stigmatellin ( approximately 2.6 x 10(6) m(-1)s(-1)) and myxothiazol ( approximately 8 x 10(5) m(-1)s(-1)), and the dissociation constant (K(d)) for quinone, were similar between pH 6.5 and 9, indicating that ligand binding is independent of the protonation state of histidine 161 of the Rieske protein (pK(a) approximately 7.6). Reduction of the Rieske protein increased the k(1) value for stigmatellin and decreased the K(d) value for quinone by 50%, without modifying the k(1) for myxothiazol. These results indicate that reduction of the Rieske protein and protonation of histidine 161 do not induce a strong stabilization of ligand binding to the quinol oxidation site, as assumed in models that propose the existence of a highly stabilized semiquinone as a reaction intermediate during quinol oxidation.     

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.     

Mechanism of ubiquinol oxidation by the bc(1) complex: different domains of the quinol binding pocket and their role in the mechanism and binding of inhibitors

Structures of mitochondrial ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) from several animal sources have provided a basis for understanding the functional mechanism at the molecular level. Using structures of the chicken complex with and without inhibitors, we analyze the effects of mutation on quinol oxidation at the Q(o) site of the complex. We suggest a mechanism for the reaction that incorporates two features revealed by the structures, a movement of the iron sulfur protein between two separate reaction domains on cytochrome c(1) and cytochrome b and a bifurcated volume for the Q(o) site. The volume identified by inhibitor binding as the Q(o) site has two domains in which inhibitors of different classes bind differentially; a domain proximal to heme b(L), where myxothiazole and beta-methoxyacrylate- (MOA-) type inhibitors bind (class II), and a distal domain close to the iron sulfur protein docking interface, where stigmatellin and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiaole (UHDBT) bind (class I). Displacement of one class of inhibitor by another is accounted for by the overlap of their volumes, since the exit tunnel to the lipid phase forces the hydrophobic "tails" to occupy common space. We conclude that the site can contain only one "tailed" occupant, either an inhibitor or a quinol or one of their reaction products. The differential sensitivity of strains with mutations in the different domains is explained by the proximity of the affected residues to the binding domains of the inhibitors. New insights into mechanism are provided by analysis of mutations that affect changes in the electron paramagnetic resonance (EPR) spectrum of the iron sulfur protein, associated with its interactions with the Q(o)-site occupant. The structures show that all interactions with the iron sulfur protein must occur at the distal position. These include interactions between quinone, or class I inhibitors, and the reduced iron sulfur protein and formation of a reaction complex between quinol and oxidized iron sulfur protein. The step with high activation energy is after formation of the reaction complex, likely in formation of the semiquinone and subsequent dissociation of the complex into products. We suggest that further progress of the reaction requires a movement of semiquinone to the proximal position, thus mapping the bifurcated reaction to the bifurcated volume. We suggest that such a movement, together with a change in conformation of the site, would remove any semiquinone formed from further interaction with the oxidized [2Fe-2S] center and also from reaction with O(2) to form superoxide anion. We also identify two separate reaction paths for exit of the two protons released in quinol oxidation.     

Site-directed mutagenesis of CCR2 identified amino acid residues in transmembrane helices 1, 2, and 7 important for MCP-1 binding and biological functions

Monocyte chemotactic protein-1 (MCP-1) binds its G-protein-coupled seven transmembrane (TM) receptor, CCR2B, and causes infiltration of monocytes/macrophages into areas of injury, infection or inflammation. To identify functionally important amino acid residues in CCR2B, we made specific mutations of nine residues selected on the basis of conservation in chemokine receptors and located TM1 (Tyr(49)), TM2 (Leu(95)), TM3 (Thr(117) and Tyr(120)), and TM7 (Ala(286), Thr(290), Glu(291), and His(297)) and in the extracellular loop 3 (Glu(278)). MCP-1 binding was drastically affected only by mutations in TM7. Reversing the charge at Glu(291) (E291K) and at His(297) (H297D) prevented MCP binding although substitution with Ala at either site had little effect, suggesting that Glu(291) and His(297) probably stabilize TM7 by their ionic interaction. E291A elicited normal Ca(2+) influx. H297A, Y49F in TM1 and L95A in TM2 that showed normal MCP-1 binding did not elicit Ca(2+) influx and elicited no adenylate cyclase inhibition at any MCP-1 concentration. MCP-1 treatment of HEK293 cells caused lamellipodia formation only when they expressed CCR2B. The mutants that showed no Ca(2+) influx and adenylate cyclase inhibition by MCP-1 treatment showed lamellipodia formation and chemotaxis. Our results show that induction of lamellipodia formation, but not Ca(2+) influx and adenylate cyclase inhibition, is necessary for chemotaxis.     

A three-pocket model for substrate coordination and selectivity by the nucleotide sugar transporters SLC35A1 and SLC35A2

The CMP-sialic acid transporter SLC35A1 and UDP-galactose transporter SLC35A2 are two well-characterized nucleotide sugar transporters with distinctive substrate specificities. Mutations in either induce congenital disorders of glycosylation. Despite the biomedical relevance, mechanisms of substrate specificity are unclear. To address this critical issue, we utilized a structure-guided mutagenesis strategy and assayed a series of SLC35A2 and SLC35A1 mutants using a rescue approach. Our results suggest that three pockets in the central cavity of each transporter provide substrate specificity. The pockets comprise (1) nucleobase (residues E52, K55, and Y214 of SLC35A1; E75, K78, N235, and G239 of SLC35A2); (2) middle (residues Q101, N102, and T260 of SLC35A1; Q125, N126, Q129, Y130, and Q278 of SLC35A2); and (3) sugar (residues K124, T128, S188, and K272 of SLC35A1; K148, T152, S213, and K297 of SLC35A2) pockets. Within these pockets, two components appear to be especially critical for substrate specificity. Y214 (for SLC35A1) and G239 (for SLC35A2) in the nucleobase pocket appear to discriminate cytosine from uracil. Furthermore, Q129 and Q278 of SLC35A2 in the middle pocket appear to interact specifically with the β-phosphate of UDP while the corresponding A105 and A253 residues in SLC35A1 do not interact with CMP, which lacks a β-phosphate. Overall, our findings contribute to a molecular understanding of substrate specificity and coordination in SLC35A1 and SLC35A2 and have important implications for the understanding and treatment of diseases associated with mutations or dysregulations of these two transporters.     

Specific triazine resistance in bacterial reaction centers induced by a single mutation in the QA protein pocket

We report here the first example of a reaction center mutant from Rhodobacter sphaeroides, where a single mutation (M266His --> Leu) taking place in the primary quinone protein pocket confers selective resistance to triazine-type inhibitors (terbutryn, ametryn, and atrazine), which bind in the secondary quinone protein pocket, at about 13 A from the mutation site. The M266His --> Leu mutation involves one of the iron atom ligands. Interestingly, neither the secondary quinone nor the highly specific inhibitor stigmatellin binding affinities are affected by the mutation. It is noticeable that in the M266His --> Ala mutant a nativelike behavior in observed. We suggest that the long side chain of Leu in position M266 may lack space to accommodate in the Q(A) pocket therefore transferring its hindrance to the Q(B) pocket. This may occur via the structural feature formed by the Q(A)-M219His-Fe-L190His-inhibitor (or Q(B)) connection, pushing L189Leu and/or L229Ile in closer contact to the triazine molecules, therefore decreasing their bindings. This opens the possibility to finely tune, in reaction center proteins, the affinity for herbicides by designing mutations distant from their binding sites.     

Zinc inhibition of bacterial cytochrome bc(1) reveals the role of cytochrome b E295 in proton release at the Q(o) site

The cytochrome (cyt) bc(1) complex (cyt bc(1)) plays a major role in the electrogenic extrusion of protons across the membrane responsible for the proton motive force to produce ATP. Proton-coupled electron transfer underlying the catalysis of cyt bc(1) is generally accepted, but the molecular basis of coupling and associated proton efflux pathway(s) remains unclear. Herein we studied Zn(2+)-induced inhibition of Rhodobacter capsulatus cyt bc(1) using enzyme kinetics, isothermal titration calorimetry (ITC), and electrochemically induced Fourier transform infrared (FTIR) difference spectroscopy with the purpose of understanding the Zn(2+) binding mechanism and its inhibitory effect on cyt bc(1) function. Analogous studies were conducted with a mutant of cyt b, E295, a residue previously proposed to bind Zn(2+) on the basis of extended X-ray absorption fine-structure spectroscopy. ITC analysis indicated that mutation of E295 to valine, a noncoordinating residue, results in a decrease in Zn(2+) binding affinity. The kinetic study showed that wild-type cyt bc(1) and its E295V mutant have similar levels of apparent K(m) values for decylbenzohydroquinone as a substrate (4.9 ± 0.2 and 3.1 ± 0.4 μM, respectively), whereas their K(I) values for Zn(2+) are 8.3 and 38.5 μM, respectively. The calorimetry-based K(D) values for the high-affinity site of cyt bc(1) are on the same order of magnitude as the K(I) values derived from the kinetic analysis. Furthermore, the FTIR signal of protonated acidic residues was perturbed in the presence of Zn(2+), whereas the E295V mutant exhibited no significant change in electrochemically induced FTIR difference spectra measured in the presence and absence of Zn(2+). Our overall results indicate that the proton-active E295 residue near the Q(o) site of cyt bc(1) can bind directly to Zn(2+), resulting in a decrease in the electron transferring activity without changing drastically the redox potentials of the cofactors of the enzyme. We conclude that E295 is involved in proton efflux coupled to electron transfer at the Q(o) site of cyt bc(1).     

Is cytochrome b glutamic acid 272 a quinol binding residue in the bc1 complex of Saccharomyces cerevisiae?

The mitochondrial bc1 complex catalyzes the oxidation of ubiquinol and the reduction of cytochrome (cyt) c coupled to a vectorial translocation of protons across the membrane. On the basis of the three-dimensional structures of the bc1 complex in the presence of the inhibitor stigmatellin, it was assumed that the substrate quinol binding involves the cyt b glutamate residue E272 and the histidine 181 on the Rieske protein. Although extensive mutagenesis of glutamate E272 has been carried out, different experimental results were recently obtained, and different conclusions were drawn to explain its role in the bifurcated electron/proton transfer at the QO site. This residue is not totally conserved during evolution. We show in this study that replacement of E272 with apolar residues proline and valine naturally present in some organisms did not abolish the bc1 activity, although it slowed down the kinetics of electron transfer. The Km value for the binding of the substrate quinol was not modified, and the EPR data showed that the quinone/quinol binding still occurred in the mutants. Binding of stigmatellin was retained; however, mutations E272P,V induced resistance toward the QO site inhibitor myxothiazol. The pH dependence of the bc1 activity was not modified in the absence of the glutamate E272. Our results suggest that this residue may not be involved in direct substrate binding or in its direct deprotonation. Revertants were selected from the respiratory deficient mutant E272P. The observed suppressor mutations introduced polar residues serine and threonine at position 272. The data lead us to suggest that E272 may be involved in a later step on the proton exit pathway via the interaction with a water molecule.