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• W2100046013 abstract "We have studied the interaction of the menaquinol analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) with dimethyl sulfoxide reductase (DmsABC) and the effect of a mutation in the DmsC subunit (DmsABCH65R) using fluorescence titration and stopped-flow methods. The titration data show that the HOQNO fluorescence is quenched when HOQNO binds to DmsABC. The binding stoichiometry is determined to be about 1:1. The mutant DmsABCH65R blocks HOQNO binding to the protein. It is therefore proposed that there is one high-affinity HOQNO binding site per DmsABC molecule located in the DmsC subunit. Stopped-flow kinetic studies show that the interaction can be described by a two-step equilibrium model, a fast bimolecular step followed by a slow unimolecular step. The quenching of HOQNO fluorescence occurs in the bimolecular step. The rates for the forward and reverse reaction of the first equilibrium are determined to be k1 = (3.9 ± 0.3) × 105m−1s−1 and k2 = 0.10 ± 0.01 s−1, respectively. The dissociation constant for the first equilibrium, Kd1 =k2/k1, is calculated to be about 260 nm. The upper limit of the overall dissociation constant is estimated to be 6 nm. We have studied the interaction of the menaquinol analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) with dimethyl sulfoxide reductase (DmsABC) and the effect of a mutation in the DmsC subunit (DmsABCH65R) using fluorescence titration and stopped-flow methods. The titration data show that the HOQNO fluorescence is quenched when HOQNO binds to DmsABC. The binding stoichiometry is determined to be about 1:1. The mutant DmsABCH65R blocks HOQNO binding to the protein. It is therefore proposed that there is one high-affinity HOQNO binding site per DmsABC molecule located in the DmsC subunit. Stopped-flow kinetic studies show that the interaction can be described by a two-step equilibrium model, a fast bimolecular step followed by a slow unimolecular step. The quenching of HOQNO fluorescence occurs in the bimolecular step. The rates for the forward and reverse reaction of the first equilibrium are determined to be k1 = (3.9 ± 0.3) × 105m−1s−1 and k2 = 0.10 ± 0.01 s−1, respectively. The dissociation constant for the first equilibrium, Kd1 =k2/k1, is calculated to be about 260 nm. The upper limit of the overall dissociation constant is estimated to be 6 nm. Escherichia coli is capable of growing anaerobically by inducing specialized respiratory chains that consist of a group of primary dehydrogenases, an intermediate electron carrier (menaquinone or ubiquinone), and a series of terminal reductases. Dimethyl sulfoxide reductase (DmsABC) 1The abbreviations used are: DmsABCdimethyl sulfoxide reductaseHOQNO2-n-heptyl-4-hydroxyquinoline-N-oxideNQNO2-n-nonyl-4-hydroxyquinoline-N-oxideMQH2menaquinolEPRelectron paramagnetic resonanceMOPS3-(N-morpholino)propanesulfonic acid. is one of the terminal reductases (1Weiner J.H. Rothery R.A. Sambasivarao D. Trieber C.A. Biochim. Biophys. Acta. 1992; 1102: 1-18Crossref PubMed Scopus (118) Google Scholar), and it catalyzes reduction of dimethyl sulfoxide and a variety of S- and N-oxides (2Simala-Grant J.L. Weiner J.H. Microbiology. 1996; 142: 3231-3239Crossref PubMed Scopus (47) Google Scholar). This membrane-associated heterotrimer contains a catalytic subunit with a molybdenum cofactor (DmsA, 87.4 kDa), an electron-transfer subunit with four [4Fe-4S] clusters (DmsB, 23.1 kDa), and a membrane-intrinsic anchor subunit (DmsC, 30.8 kDa) (3Bilous P.T. Cole S.T. Anderson W.F. Weiner J.H. Mol. Microbiol. 1988; 2: 785-795Crossref PubMed Scopus (128) Google Scholar). dimethyl sulfoxide reductase 2-n-heptyl-4-hydroxyquinoline-N-oxide 2-n-nonyl-4-hydroxyquinoline-N-oxide menaquinol electron paramagnetic resonance 3-(N-morpholino)propanesulfonic acid. DmsC is a hydrophobic polypeptide that traverses eight times and anchors the DmsAB dimer to the membrane (4Weiner J.H. Shaw G. Turner R.J. Trieber C.A. J. Biol. Chem. 1993; 268: 3238-3244Abstract Full Text PDF PubMed Google Scholar). It is involved in menaquinol (MQH2) binding and oxidation, and in electron transport. DmsABC is able to draw electrons from the menaquinol pool to DmsC and transfer them through DmsB to the catalytic subunit, DmsA. In a recent electron paramagnetic resonance (EPR) study (5Rothery R.A. Weiner J.H. Biochemistry. 1996; 35: 3247-3257Crossref PubMed Scopus (61) Google Scholar), it was shown that DmsCH65 is involved in MQH2 binding and oxidation. It was suggested that a mutation of the DmsC subunit, DmsCH65R, may block binding of the MQH2 analog HOQNO to the protein. However, there was no direct observation available about this possible blockage of HOQNO binding. For another terminal reductase, fumarate reductase, it has been reported that there are two separate MQH2 binding sites located in subunits C and D of the enzyme, respectively (6Westenberg D.J. Gunsalus R.P. Ackrell B.A.C. Cecchini G. J. Biol. Chem. 1990; 256: 19560-19567Abstract Full Text PDF Google Scholar, 7Westenberg D.J. Gunsalus R.P. Ackrell B.A.C. Sices H. Cecchini G. J. Biol. Chem. 1993; 268: 815-822Abstract Full Text PDF PubMed Google Scholar, 8Cecchini G. Sices H. Schroder I. Gunsalus R.P. J. Bacteriol. 1995; 177: 4587-4592Crossref PubMed Google Scholar). The MQH2 binding affinity of the site in subunit D is higher than that of the site in subunit C. For DmsABC, however, the number and the location of MQH2 binding site(s) have not been determined. HOQNO is a structural analog of MQH2, and it is known as a classical inhibitor of the mitochondrial cytochrome creductase. An earlier study showed that the fluorescence of HOQNO was completely quenched by binding to submitochondrial particles of beef heart (9van Ark G. Berden J.A. Biochim. Biophys. Acta. 1977; 459: 119-137Crossref PubMed Scopus (101) Google Scholar). It was concluded that the inhibition of electron transfer by HOQNO was caused by binding to the specific binding site. Fluorescence quenching was also observed when an HOQNO analog, NQNO, bound to the mitochondrial cytochrome c reductase (10Brandt U. von Jagow G. Eur. J. Biochem. 1991; 195: 163-170Crossref PubMed Scopus (76) Google Scholar). NQNO is a specific inhibitor of the inner facing quinone-reaction center (Qi) of the mitochondrial cytochrome creductase. The fluorescence of HOQNO has provided a convenient means for studying HOQNO binding and inhibition reactions. It is expected, therefore, that a fluorescence spectroscopic study of the interaction of HOQNO with DmsABC could provide useful information for determining the number and the location of MQH2 binding site(s) in DmsABC. In this paper, we examine the binding stoichiometry and kinetics for the interaction of HOQNO with DmsABC using fluorescence titration and stopped-flow methods. We show that HOQNO binds to DmsABC with approximately 1:1 stoichiometry and the interaction can be described by a two-step equilibrium model. We also provide evidence that a mutation of the DmsC subunit, DmsABCH65R, blocks the binding of the MQH2 analog HOQNO to the protein as suggested recently in an EPR study (5Rothery R.A. Weiner J.H. Biochemistry. 1996; 35: 3247-3257Crossref PubMed Scopus (61) Google Scholar). E. coli HB101 [supE44 hsdS20 2(rB− mB− ) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1] (11Boyer H.W. Roulland-Dussoix D. J. Mol. Biol. 1969; 41: 459-472Crossref PubMed Scopus (2571) Google Scholar) was used for expression of wild-type and mutant DmsABC. For overexpression of DmsABC and DmsABCH65R, bacteria were transformed with pDMS160 and pDMS160-H65R, respectively (5Rothery R.A. Weiner J.H. Biochemistry. 1996; 35: 3247-3257Crossref PubMed Scopus (61) Google Scholar, 12Rothery R.A. Weiner J.H. Biochemistry. 1991; 30: 8296-8305Crossref PubMed Scopus (90) Google Scholar, 13Trieber C.A. Rothery R.A. Weiner J.H. J. Biol. Chem. 1994; 269: 7103-7109Abstract Full Text PDF PubMed Google Scholar). All manipulations of strains were carried out as described by Sambrook et al. (14Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Cells were grown anaerobically in 20-liter batches at 37 °C on a glycerol-fumarate minimal medium for 48 h as described previously (15Bilous P.T. Weiner J.H. J. Bacteriol. 1985; 162: 1151-1155Crossref PubMed Google Scholar). Cells were harvested using a Pellicon membrane system (16Weiner J.H. MacIssac D.P. Bishop R.E. Bilous P.T. J. Bacteriol. 1988; 170: 1505-1510Crossref PubMed Google Scholar), washed, lysed by passage through a French pressure cell, and subjected to differential centrifugation. The isolated membranes were resuspended in 100 mm MOPS and 5 mm EDTA (pH 7.0), frozen in liquid nitrogen, and then stored at −70 °C until use (12Rothery R.A. Weiner J.H. Biochemistry. 1991; 30: 8296-8305Crossref PubMed Scopus (90) Google Scholar). Protein concentrations were determined by a modified Lowry assay in the presence of 1% SDS using a Bio-Rad serum albumin protein standard (17Markwell M.A.D. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5328) Google Scholar). The concentration of DmsABC was calculated from the total concentration of [4Fe-4S] clusters measured by the EPR spin quantitation and by assuming that there are four [4Fe-4S] clusters per DmsABC molecule (18Paulsen K.E. Stankovich M.T. Orville A.M. Methods Enzymol. 1993; 227: 396-411Crossref PubMed Scopus (18) Google Scholar, 19Cammack R. Weiner J.H. Biochemistry. 1990; 29: 8410-8416Crossref PubMed Scopus (77) Google Scholar). HOQNO was obtained from Sigma. The concentration of HOQNO was determined spectrophotometrically after diluting the ethanolic stock solution in 1 mm sodium hydroxide using an extinction coefficient of 9450m−1 cm−1 at 346 nm (20Cornforth J.W. James A.T. Biochem. J. 1956; 63: 124-130Crossref PubMed Scopus (73) Google Scholar). Fluorescence measurements were carried out using a Perkin-Elmer LS-50B luminescence spectrometer. The fluorescence emission spectrum of HOQNO in 100 mm MOPS and 5 mm EDTA (pH 7.0) exhibits a maximum at 479 nm with the excitation at 341 nm (data not shown). In fluorescence titration experiments, aliquots of a 50 μmHOQNO stock solution were added to the cuvette containing the protein in 100 mm MOPS and 5 mm EDTA (pH 7.0), and fluorescence emissions were measured at 479 nm (with excitation at 341 nm). The background fluorescence of the protein sample in the absence of HOQNO was subtracted from the fluorescence of the sample in the presence of HOQNO. The stopped-flow experiments were performed using a Sequential Bio SX-17MV stopped-flow spectrofluorimeter (Applied Photophysics Ltd., Leatherhead, UK). In a typical experiment, DmsABC or DmsABCH65R (1 or 2 μm) in 100 mm MOPS and 5 mm EDTA (pH 7.0) was mixed with an equal volume of various concentrations of HOQNO (1–14 μm) in the same buffer. Temperature was maintained at 25 °C. The fluorescence of HOQNO was excited at 341 nm, and the emissions above 400 nm were recorded through a cut off filter SGG-400-1.00 (CVI Laser Co., Albuquerque, NM). For each concentration of HOQNO, at least three runs were performed and 1000 or 2000 data points were collected. After averaging, data were fitted to an appropriate equation using the software supplied by Applied Photophysics. Under our experimental conditions, quenching of HOQNO fluorescence by binding to DmsABC was biphasic, and the observed fluorescence, F, was best fitted to a double exponential equation, F=Afe−kft+Ase−kst+bEquation 1 where Af and As are the amplitudes of the fast and slow phase, and kf andks are the observed rates for the fast and slow phase, respectively, t is time and b is an off set value of the stopped-flow instrument. The interaction of HOQNO with DmsABC can be described by the following model (21Halford S.E. Biochem. J. 1975; 149: 411-422Crossref PubMed Scopus (33) Google Scholar, 22Hiromi K. Kinetics of Fast Enzyme Reactions. Halsted Press, New York1979: 187-286Google Scholar, 23Hammes G.G. Enzyme Catalysis and Regulation. Academic Press, New York1982: 61-75Crossref Google Scholar) (see “Discussion”), E+L⇌k2k1EL⇌k4k3EL*Equation 2 where E represents DmsABC; L, the ligand (HOQNO); EL, the initial complex of DmsABC with HOQNO before the isomerization takes place; EL*, the final complex product; k1 and k3 are the rate constants for the forward reactions inm−1 s−1 and s−1, respectively, and k2 andk4 are the rate constants for the reverse reactions in s−1. In the case where the bimolecular process is much faster than the unimolecular process and the initial concentration of L is much higher than the concentration ofE, the observed first-order rates for the fast phase (kf) and the slow phase (ks) are given by Equations 3 and 4, respectively, kf=k2+k1[L]Equation 3 ks=k4+k3/1+Kd1/[L]Equation 4 where Kd1 =k2/k1 is the dissociation constant of the first equilibrium in Equation 2. Under our experimental conditions, the plot of HOQNO fluorescence against the concentration of HOQNO was linear up to 7 μmof HOQNO (after mixing) (data not shown). This was, therefore, the highest concentration of HOQNO used in this work. Toward the low end of the HOQNO concentrations used herein, the condition that initial concentration of L is much higher than the concentration ofE (0.5 μm after mixing) does not hold. However, the observed first-order rates for the fast phase,kf, can still be analyzed using Equation 3 as demonstrated by Halford (21Halford S.E. Biochem. J. 1975; 149: 411-422Crossref PubMed Scopus (33) Google Scholar). From the fit of the observed kf data to Equation 3,k1 and k2 can be obtained. Under our experimental conditions, deviations of the observedks were too large to be fitted to Equation 4 to yield reliable k3 and k4. To evaluate the values of k3 andk4, another approach using the Glint program (Applied Photophysics) was employed. This program enables one to globally analyze a complete data set measured at all wavelengths according to a proposed reaction scheme and to obtain the reaction parameters from the best fit of the calculated data to the experimental data. Kinetic data irrelevant of wavelength (the case of this work) or measured at a single wavelength can also be analyzed using this program. In this work, using the reaction scheme (Equation 2), and thek1 and k2 determined from Equation 3, kinetic traces measured at various HOQNO concentrations were analyzed using the Glint program, and the values ofk3 and k4 were evaluated from the good fits of the calculated kinetic traces to the observed traces. The binding of HOQNO to DmsABC was examined by measuring the HOQNO fluorescence emission (at 479 nm with excitation at 341 nm) in the absence and in the presence of DmsABC. In the absence of DmsABC, the plot of fluorescence measured by titrating the buffer (100 mm MOPS and 5 mmEDTA, pH 7.0) against the concentration of HOQNO gave a straight line with a positive slope as shown in Fig.1 A. This fluorescence was attributed to free HOQNO molecules (not bound to other compounds). In the presence of DmsABC, however, the initial portion of each titration curve was flat (Fig. 1 A), indicating that there was no increase of fluorescence intensity upon the addition of a certain concentration of HOQNO to the protein. In other words, there was no free HOQNO present in the system to emit fluorescence. It was clear that the fluorescence of HOQNO was quenched in the presence of DmsABC and this quenching was because of HOQNO binding to DmsABC. Further addition of HOQNO to the protein caused a sudden increase of fluorescence intensity, suggesting that free HOQNO became available when the amount of HOQNO added to the protein was higher than a certain concentration at a given concentration of DmsABC. It was not possible to construct a Scatchard plot from the titration data in Fig. 1 A because of the sharp transition of the titration curves. To obtain the stoichiometry of HOQNO binding to DmsABC, four concentrations of DmsABC (0.25, 0.5, 0.75, and 1.0 μm) were titrated with HOQNO in 100 mm MOPS and 5 mm EDTA at pH 7.0 (Fig. 1 A). The extrapolations of the linear portions of the titration curves to zero fluorescence yield four intercepts on the x-axis of the plot. These intercepts represent the concentrations of HOQNO bound to DmsABC at the given concentrations of DmsABC. Therefore, from the plot of the intercepts against the concentration of DmsABC, Fig.1 B, the stoichiometry of HOQNO binding to DmsABC can be determined to be approximately 1:1. It has been suggested in a recent EPR study (5Rothery R.A. Weiner J.H. Biochemistry. 1996; 35: 3247-3257Crossref PubMed Scopus (61) Google Scholar) that residue His-65 in the DmsC subunit may be involved in MQH2binding and a His to Arg mutation, DmsABCH65R, may block binding the MQH2 analog HOQNO to the protein. To verify this, titrations of 0.4 μm wild-type DmsABC and mutant DmsABCH65R with HOQNO in 100 mm MOPS and 5 mm EDTA (pH 7.0) were carried out and the data obtained were compared as shown in Fig. 2. For the wild-type protein, the initial portion of the titration curve was flat indicating quenching of HOQNO fluorescence because of HOQNO binding to the protein. Further increase of HOQNO concentration caused a sharp increase of fluorescence intensity. For the mutant DmsABCH65R, however, the titration data gave a straight line with a positive slope, indicating that there was no quenching of HOQNO fluorescence. In other words, in the case of titration of DmsABCH65R, the HOQNO added to the protein was in the free form and not bound to the protein. Fig. 2, thus, demonstrates that the mutation of DmsABCH65R blocks HOQNO binding to the protein. To investigate the kinetics and mechanism of the interaction of HOQNO with DmsABC, the stopped-flow fast kinetic technique was employed. Fig.3 shows a plot of the observed fluorescence intensities against the concentration of HOQNO obtained by mixing various concentrations of HOQNO in buffer of 100 mmMOPS and 5 mm EDTA (pH 7.0) with the same buffer or with 2 μm wild-type DmsABC (in the buffer). After mixing the protein with HOQNO, a decrease of fluorescence intensity with time was observed as shown in the inset of Fig. 3 indicating quenching of HOQNO fluorescence. The magnitude of the initial fluorescence intensity measured immediately after mixing HOQNO with DmsABC was very similar to the magnitude of the fluorescence intensity measured in the absence of the protein, indicating that no detectable reaction occurred during the dead time of the stopped-flow instrument. The same results were also obtained by mixing 1 μm of DmsABC with various concentrations of HOQNO in 100 mm MOPS and 5 mmEDTA (pH 7.0) under the conditions in Figs. 5 B and 6 (data not shown). On the other hand, for the final fluorescence intensity measured 50 s after mixing, the initial portion of the curve was flat and the intercept on the x-axis (about 1 μm) obtained by extrapolating the linear portion of the curve to zero fluorescence was in good agreement with the 1:1 binding stoichiometry obtained under the steady-state conditions (Figs. 1 and2). Fig. 3 demonstrates that the fluorescence quenching observed after mixing HOQNO with DmsABC is because of HOQNO binding to the protein.Figure 5Kinetic analysis of fluorescence quenching because of the interaction of HOQNO with DmsABC. A,quenching of HOQNO fluorescence observed by mixing 1 μmof DmsABC with 2 μm HOQNO in 100 mm MOPS and 5 mm EDTA at pH 7.0 (25 °C) and the fit of these data to Equation 1. From the fit, the observed first-order rates and the amplitudes for the fast and slow phase were determined to bekf = 0.52 ± 0.06 s−1,ks = 0.07 ± 0.008 s−1,Af = 0.041 ± 0.003, and As= 0.035 ± 0.003, respectively. The lower part of the figure shows the residuals of the fit. B, effect of HOQNO concentration on kf. The kf data were determined in the same way as described in panel A,except varying HOQNO concentrations. The solid linerepresents the fit of the kf data to Equation 3, from which the rate constants for the first equilibrium in Equation 2were determined to be k1 = (3.9 ± 0.3) × 105m−1 s−1 andk2 = 0.10 ± 0.01 s−1, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Dependence ofAf andAs on HOQNO concentration.The amplitudes for the fast phaseAf (•) and slow phase As (○) were obtained by mixing 1 μm of DmsABC with various concentrations of HOQNO in 100 mm MOPS and 5 mmEDTA at pH 7.0 (25 °C) and by fitting the observed traces to Equation 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The DmsABCH65R mutant was used as a non-HOQNO binding control for this work. Fig. 4 shows the plot of the fluorescence intensities against the concentration of HOQNO obtained by mixing 2 μm mutant DmsABCH65Rwith various concentrations of HOQNO in 100 mm MOPS and 5 mm EDTA (pH 7.0). In contrast to Fig. 3, the plot of the initial fluorescence intensity (measured immediately after mixing) and the final fluorescence intensity (measured 50 s after mixing) against the concentration of HOQNO gave two straight lines that not only had the same slope but also overlapped each other within experimental error. Clearly, there was no significant quenching of HOQNO fluorescence in this case. Fig. 4 indicates that the mutation of DmsABCH65R blocks HOQNO binding to the protein, which agrees with the steady-state titration data (Fig. 2). Furthermore, this result serves as a good control for the stopped-flow experiment, confirming that the nonlinear feature of the curve for the final fluorescence intensity in Fig. 3 is not because of an artifact. As shown in Fig. 3, binding HOQNO to DmsABC causes a quenching of HOQNO fluorescence, and this quenching process can be followed using the stopped-flow method. To further investigate the kinetics of HOQNO binding to DmsABC, 1 μm DmsABC was rapidly mixed with various concentrations of HOQNO using the stopped-flow method. Fig.5 A shows a typical trace of fluorescence quenching observed after mixing 1 μm of DmsABC with 2 μm HOQNO in 100 mm MOPS and 5 mm EDTA at pH 7.0 (25 °C) and the residuals for fitting these data to Equation 1. The fluorescence quenching process was completed about 50 s after mixing. The observed quenching trace had two phases, a fast phase followed by a slow phase, and it was best fitted with the double exponential equation, Equation 1. This was also true at other HOQNO concentrations used in this work (Fig.5 B). From the fit, the observed first-order rates and the amplitudes for the fast and slow phase at the given concentration of HOQNO, kf, ks,Af, and As, can be determined. The first-order rate for the fast phase (kf) observed by mixing 1 μm DmsABC with various concentrations of HOQNO in 100 mm MOPS and 5 mmEDTA at pH 7.0 (25 °C) was plotted against the concentration of HOQNO and the data obtained were fitted to Equation 3 as shown in Fig.5 B, from which the rate constants for the forward and reverse reactions of the first equilibrium in Equation 2 were determined to be k1 = (3.9 ± 0.3) × 105m−1 s−1 andk2 = 0.10 ± 0.01 s−1respectively. Thus the dissociation constant for the first equilibrium,Kd1 =k2/k1, was calculated to be about 260 nm. The rate constants for the forward and reverse reactions of the second equilibrium in Equation 2, k3 andk4, were evaluated with the Glint program (Applied Photophysics). Using the reaction scheme described in Equation2, and the k1 (=3.9 × 105m−1 s−1) andk2 (= 0.10 s−1) determined from the fit as shown in Fig. 5 B, k3 = 0.40 ± 0.04 s−1 and k4 ≤ 0.01 s−1 were obtained from the good fits of the calculated kinetic traces to the observed traces (data not shown). Therefore, the association constant of the second equilibrium,Ka2 =k3/k4 ≥ 40, was obtained. The upper limit of the overall dissociation constant for Equation 2 was thus estimated to be about 6 nm,i.e. Kdoverall =Kd1/(1 +Ka2) ≤ 6 nm. To investigate the effect of the ligand (HOQNO) concentration on the kinetics of HOQNO binding to DmsABC, the amplitudes for the fast phase (Af) and the slow phase (As) were plotted against the concentration of HOQNO (Fig.6). The Af andAs were obtained by mixing 1 μm DmsABC with various concentrations of HOQNO in 100 mm MOPS and 5 mm EDTA at pH 7.0 (25 °C) and by fitting the observed traces with Equation 1. The amplitude for the fast phase increased with the increase of HOQNO concentration up to about 4 μm then became saturated, whereas the amplitude for the slow phase increased initially with HOQNO concentration up to 1 μm and then it started to decrease with the increase of HOQNO concentration. In bacterial anaerobic electron transport menaquinol shuttles reducing equivalents from the primary dehydrogenases to the terminal reductases within the cytoplasmic membrane. However, relatively little is known about the interaction of menaquinol with these enzymes. In this study, we have utilized direct steady state and rapid reaction methodologies to investigate the binding of a fluorescent analog of menaquinol to DmsABC. We have shown that HOQNO interacts with very high affinity and 1:1 stoichiometry at a site within the DmsC subunit. Mutation of His-65 to Arg in DmsC blocks quinol activity and eliminates HOQNO binding. Our finding of only one exchangeable binding site, by direct measurement, differs from studies with fumarate reductase (6Westenberg D.J. Gunsalus R.P. Ackrell B.A.C. Cecchini G. J. Biol. Chem. 1990; 256: 19560-19567Abstract Full Text PDF Google Scholar, 7Westenberg D.J. Gunsalus R.P. Ackrell B.A.C. Sices H. Cecchini G. J. Biol. Chem. 1993; 268: 815-822Abstract Full Text PDF PubMed Google Scholar, 8Cecchini G. Sices H. Schroder I. Gunsalus R.P. J. Bacteriol. 1995; 177: 4587-4592Crossref PubMed Google Scholar) and nitrate reductase (24Giordani R. Buc J. Cornish-Bowden A. Cardenas M.L. Eur. J. Biochem. 1997; 250: 567-577Crossref PubMed Scopus (31) Google Scholar), where two quinol binding sites have been proposed by indirect methods. In the stopped-flow studies, the biphasic nature of the observed quenching process and the nonlinear increase of the observed slow rate (ks) with HOQNO concentration reaching a plateau at higher concentrations of HOQNO (data not shown) suggest that the two-step mechanism (Equation 2) (21Halford S.E. Biochem. J. 1975; 149: 411-422Crossref PubMed Scopus (33) Google Scholar, 22Hiromi K. Kinetics of Fast Enzyme Reactions. Halsted Press, New York1979: 187-286Google Scholar, 23Hammes G.G. Enzyme Catalysis and Regulation. Academic Press, New York1982: 61-75Crossref Google Scholar), a fast bimolecular process followed by a slow unimolecular process, can be applied to describe the quenching process resulting from binding HOQNO to DmsABC. In this mechanism, it is assumed that a loosely bound complex of the HOQNO (L) with DmsABC (E), EL, is formed rapidly by a bimolecular association and then it is converted to a more tightly and specifically bound complex, EL*, by a slow unimolecular process (isomerization). The isomerization may take place through several possible pathways, such as rearrangement ofEL without any conformational change in E orL, a conformational change in either E orL, or conformational changes in both E andL (22Hiromi K. Kinetics of Fast Enzyme Reactions. Halsted Press, New York1979: 187-286Google Scholar). It is expected that stopped-flow kinetic studies can provide some useful information about the bimolecular association of HOQNO with DmsABC and the subsequent isomerization process. The effect of HOQNO concentration on the amplitudes of the fast and slow phase may be interpreted as follows. When the concentration of HOQNO is lower than or comparable with the dissociation constant of the first equilibrium in Equation 2, Kd1, but higher than the overall dissociation constant, only a small amount of protein is associated with the ligand (HOQNO) to form the complex EL by the fast bimolecular process, which is observed as a fast phase of quenching. The subsequent slow isomerization process of convertingEL to EL* would shift the first equilibrium in Equation 2 to the right-hand side slowly. This slow shift of the first equilibrium is observed as a subsequent slow phase of quenching. Alternatively, when the concentration of HOQNO is increased to higher than Kd1, more and more DmsABC molecules are associated with HOQNO to give EL through the fast bimolecular process, thus, less and less free DmsABC molecules are available for the subsequent shift of the first equilibrium to the right-hand side caused by the slow isomerization process. Therefore, the amplitude of the observed fast phase of quenching increases with increase of HOQNO concentration until becoming saturated, whereas the observed slow phase of quenching decreases with increase of HOQNO concentration, and this would finally result in that the amplitude of the slow phase would be negligible compared with the amplitude of the fast phase. These results suggest that the quenching of HOQNO fluorescence occurs in the bimolecular step of the association ofE with L rather than in the unimolecular step of forming EL* (see Equation 2). Primary dehydrogenases and terminal reductases have been the subject of a large number of steady-state kinetic investigations (see Refs. 24Giordani R. Buc J. Cornish-Bowden A. Cardenas M.L. Eur. J. Biochem. 1997; 250: 567-577Crossref PubMed Scopus (31) Google Scholar, 25Shiraishi F. Savageau M.A. J. Biol. Chem. 1992; 267: 22934-22943Abstract Full Text PDF PubMed Google Scholar, 26Kotik M. Zuber H. 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Chem. 1998; 273: 10851-10856Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholaras examples), but rapid reaction methodologies have been limited because of the lack of available probes. We have now shown that fluorescent HOQNO can be used to monitor the quinol binding reaction. Although the steady-state methodology is useful for stoichiometry, the affinity is so tight that Scatchard plots are not useful. The stopped-flow method allows determination of rates and dissociation constants. Although the three-dimensional structures of several quinone-binding proteins have been determined (32Roy C. Lancaster D. Michel H. Structure ( Lond. ). 1997; 5: 1339-1359Abstract Full Text Full Text PDF PubMed Google Scholar, 33Xia D. Yu C.-A. Kim H. Xia J.-Z. Kachurin A.M. Zhang L. Yu L. Deisenhofer J. Science. 1997; 277: 60-66Crossref PubMed Scopus (870) Google Scholar), it is not yet possible to define a quinone binding motif. This technique will prove useful in examining site-directed mutations in the quinol binding region or in conformationally coupled changes that alter quinone binding. This work and a recent study on the interaction of HOQNO with fumarate reductase using EPR and steady-state fluorescence spectroscopic methods (34Rothery R.A. Weiner J.H. Eur. J. Biochem. 1998; 254: 588-595Crossref PubMed Scopus (25) Google Scholar) demonstrate the utility of the characteristics of HOQNO fluorescence and its structural analogy to MQH2 for studying interactions of MQH2 with terminal reductases in respiratory chains. Studies on the interactions of HOQNO with E. coli. nitrate reductase 2R. A. Rothery, F. Blasco, and J. H. Weiner, unpublished results. and with fumarate reductase 3Z. Zhao, R. A. Rothery, and J. H. Weiner, unpublished results. are in progress. We thank Dr. R. A. Rothery for helpful discussions, assisting in protein preparations, and critically reading the manuscript, and Dr. H. B. Dunford in the Department of Chemistry, University of Alberta for providing the stopped-flow facility." @default.
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