Evidence for a novel mechanism of time-resolved flavin fluorescence depolarization in glutathione reductase

Time-resolved flavin fluorescence anisotropy studies on glutathione reductase (GR) have revealed a remarkable new phenomenon: wild-type GR displays a rapid process of fluorescence depolarization, that is absent in mutant enzymes lacking a nearby tyrosine residue that blocks the NADPH-binding cleft. Fluorescence lifetime data, however, have shown a more rigid active-site structure for wild-type GR than for the tyrosine mutants. These results suggest that the rapid depolarization in wild-type GR originates from an interaction with the flavin-shielding tyrosine, and not from restricted reorientational motion of the flavin. A novel mechanism of fluorescence depolarization is proposed that involves a transient charge-transfer complex between the tyrosine and the light-excited flavin, with a concomitant change in the direction of the emission dipole moment of the flavin. This interaction is likely to result from side-chain relaxation of the tyrosine in the minor fraction of enzyme molecules in which this residue is in an unsuitable position for immediate fluorescence quenching at the moment of excitation. Support for this mechanism is provided by binding studies with NADP+ and 2'P-5'ADP-ribose that can intercalate between the flavin and tyrosine and/or block the latter. Fluorescence depolarization analyses as a function of temperature and viscosity confirm the dynamic nature of the process. A comparison with fluorescence depolarization effects in a related flavoenzyme indicates that this mechanism of flavin fluorescence depolarization is more generally applicable.     

Time-resolved spectroscopic studies of the AppA blue-light receptor BLUF domain from Rhodobacter sphaeroides

AppA is a blue-light and redox-responding regulator of photosynthesis gene expression in Rhodobacter sphaeroides. Detailed time-resolved fluorescence spectroscopy and subpicosecond transient absorption spectroscopy study of the BLUF domain is presented for wild-type AppA (AppAwt) and a photoinactive Y21F mutant of AppA. The main findings discussed here are that (1) time-resolved laser excitation studies on dark-adapted protein show that AppAwt and Y21F mutant protein exhibits a fluorescence decay with a lifetime of 0.6 ns. Dark-adapted AppAwt but not Y21F also exhibits slower fluorescence decay with a lifetime of 1.7 ns. Analysis of AppAwt that was light-excited to a stable light-adapted form prior to data collection shows monoexponential fluorescence decay with a lifetime of 1.0 ns. This component disappeared after 1 min of data collection after which the original "dark-adapted" values were recovered, demonstrating the presence of a approximately 1 min lifetime intermediate during the return of AppA from light- to dark-adapted form. (2) Transient absorption spectral analysis reveals a very fast rising of transient absorption (<1 ps) for AppAwt. This fast component is missing in the Y21F mutant, which lacks Tyr21, giving rise to a slower transient absorption at 4-6 ps. In the AppAwt transient spectra, most ground states recover within approximately 30 ps, compared to approximately 90-130 ps in the mutant Y21F. We propose that a temporary electron transfer occurs from Tyr21 to the N5 of flavin in AppAwt and is a triggering event for subsequent hydrogen-bond rearrangements. Dynamics of the AppA photocycle is discussed in view of the currently solved crystallographic structure of AppA.     

Switching kinetic mechanism and putative proton donor by directed mutagenesis of glutathione reductase

By directed mutagenesis of the cloned Escherichia coli gor gene encoding the flavoprotein glutathione reductase, Tyr-177 (the residue corresponding to Tyr-197 in the NADPH-binding pocket of the homologous human enzyme) was changed to phenylalanine (Y177F), serine (Y177S), and glycine (Y177G). The catalytic activity of the Y177F mutant was very similar to that of the wild-type enzyme, but that of the Y177S and Y177G mutants was substantially diminished. However, all three mutants retained the ability to protect the reduced flavin from adventitious oxidation, indicating that Tyr-177 does not act as a simple "lid" on the NADPH-binding pocket and that the protection of the reduced enzyme must be due largely to burial of the isoalloxazine ring in the protein. The wild-type enzyme and Y177F mutant displayed ping-pong kinetics, but the Y177S and Y177G mutants appeared to have switched to an ordered sequential mechanism. This could be explained by supposing that the enzyme normally functions by a hybrid kinetic mechanism and that the Y177S and Y177G mutations diverted flux from the ping-pong loop favored by the wild-type enzyme to an ordered sequential loop. The necessary change in the partitioning of the common E-NADPH intermediate could be caused by a slowing of the formation of the EH2 intermediate on the ping-pong loop, or by the observed concomitant fall in the Km for glutathione favoring flux through the ordered sequential loop. In another experiment, His-439, thought to act as a proton donor/acceptor in the glutathione-binding pocket, was mutated to a glutamine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome b5 reductase: role of the si-face residues, proline 92 and tyrosine 93, in structure and catalysis

The conserved sequence motif "RxY(T)(S)xx(S)(N)" coordinates flavin binding in NADH:cytochrome b(5) reductase (cb(5)r) and other members of the flavin transhydrogenase superfamily of oxidoreductases. To investigate the roles of Y93, the third and only aromatic residue of the "RxY(T)(S)xx(S)(N)" motif, that stacks against the si-face of the flavin isoalloxazine ring, and P92, the second residue in the motif that is also in close proximity to the FAD moiety, a series of rat cb(5)r variants were produced with substitutions at either P92 or Y93, respectively. The proline mutants P92A, G, and S together with the tyrosine mutants Y93A, D, F, H, S, and W were recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein was found to bind FAD in a 1:1 cofactor:protein stoichiometry while UV CD spectra suggested similar secondary structure organization among all nine variants. The tyrosine variants Y93A, D, F, H, and S exhibited varying degrees of blue-shift in the flavin visible absorption maxima while visible CD spectra of the Y93A, D, H, S, and W mutants exhibited similar blue-shifted maxima together with changes in absorption intensity. Intrinsic flavin fluorescence was quenched in the wild type, P92S and A, and Y93H and W mutants while Y93A, D, F, and S mutants exhibited increased fluorescence when compared to free FAD. The tyrosine variants Y93A, D, F, and S also exhibited greater thermolability of FAD binding. The specificity constant (k(cat)/K(m)(NADH)) for NADH:FR activity decreased in the order wild type > P92S > P92A > P92G > Y93F > Y93S > Y93A > Y93D > Y93H > Y93W with the Y93W variant retaining only 0.5% of wild-type efficiency. Both K(s)(H4NAD) and K(s)(NAD+) values suggested that Y93A, F, and W mutants had compromised NADH and NAD(+) binding. Thermodynamic measurements of the midpoint potential (E degrees ', n = 2) of the FAD/FADH(2) redox couple revealed that the potentials of the Y93A and S variants were approximately 30 mV more positive than that of wild-type cb(5)r (E degrees ' = -268 mV) while that of Y93H was approximately 30 mV more negative. These results indicate that neither P92 nor Y93 are critical for flavin incorporation in cb(5)r and that an aromatic side chain is not essential at position 93, but they demonstrate that Y93 forms contacts with the FAD that effectively modulate the spectroscopic, catalytic, and thermodynamic properties of the bound cofactor.     

Conformational dynamics and intersubunit energy transfer in wild-type and mutant lipoamide dehydrogenase from Azotobacter vinelandii. A multidimensional time-resolved polarized fluorescence study.

Time-resolved fluorescence and fluorescence anisotropy data surfaces of flavin adenine dinucleotide bound to lipoamide dehydrogenase from Azotobacter vinelandii in 80% glycerol have been obtained by variation of excitation energy and temperature between 203 and 303 K. The fluorescence kinetics of a deletion mutant lacking 14 COOH-terminal amino acids were compared with the wild-type enzyme to study a possible interaction of the COOH-terminal tail with the active site of the enzyme. The flavin adenine dinucleotide fluorescence in both proteins exhibits a bimodal lifetime distribution as recovered by the maximum entropy method of data analysis. The difference in standard enthalpy and entropy of associated conformational substates was retrieved from the fractional contributions of the two lifetime classes. Activation energies of thermal quenching were obtained that confirm that the isoalloxazines in the deletion mutant are solvent accessible in contrast to the wild-type enzyme. Red-edge spectroscopy in conjunction with variation of temperature provides the necessary experimental axes to interpret the fluorescence depolarization in terms of intersubunit energy transfer rather than reorientational dynamics of the flavins. The results can be explained by a compartmental model that describes the anisotropy decay of a binary, inhomogeneously broadened, homoenergy transfer system. By using this model in a global analysis of the fluorescence anisotropy decay surface, the distance between and relative orientation of the two isoalloxazine rings are elucidated. For the wild-type enzyme, this geometrical information is in agreement with crystallographic data of the A. vinelandii enzyme, whereas the mutual orientation of the subunits in the deletion mutant is slightly altered. In addition, the ambiguity in the direction of the emission transition moment in the isoalloxazine ring is solved. The anisotropy decay parameters also provide information on electronic and dipolar relaxational properties of the flavin active site. The local environment of the prosthetic groups in the deletion mutant of the A. vinelandii enzyme is highly inhomogeneous, and a transition from slow to rapid dipolar relaxation is observed over the measured temperature range. In the highly homogeneous active site of the wild-type enzyme, dipolar relaxation is slowed down beyond the time scale of fluorescence emission at any temperature studied. Our results are in favor of a COOH-terminal polypeptide interacting with the active site, thereby shielding the isoalloxazines from the solvent. This biological system forms a very appropriate tool to test the validity of photophysical models describing homoenergy transfer.     

Mutagenesis of the redox-active disulfide in mercuric ion reductase: catalysis by mutant enzymes restricted to flavin redox chemistry

Mercuric reductase, a flavoenzyme that possess a redox-active cystine, Cys135Cys140, catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, we have constructed mutants lacking a redox-active disulfide by eliminating Cys135 (Ala135Cys140), Cys140 (Cys135Ala140), or both (Ala135Ala140). Additionally, we have made double mutants that lack Cys135 (Ala135Cys139Cys140) or Cys140 (Cys135Cys139Ala140) but introduce a new Cys in place of Gly139 with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH2 redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. These differences are manifested in a 23-nm range in enzyme-bound FAD lambda max values, an 80-nm range in thiolate to flavin charge-transfer absorbance maxima, and a ca. 100-mV range in FAD reduction potential. Preliminary evidence for the Ala135Cys139Cys140 mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala135Cys140 protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. For these activities, there is a linear correlation between log kappa cat and enzyme-bound FAD reduction potential. In a sensitive Hg(II)-mediated enzyme-bound FADH2 reoxidation assay, all mutant enzymes were able to undergo at least one catalytic event at rates 50-1000-fold slower than that of the wild-type enzyme. We have also observed the reduction of Hg(II) by free FADH2. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. We conclude that the Cys135 and Cys140 thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate.     

Kinetic studies on the role of Lys-171 and Lys-358 in the beta subunit of sarcosine oxidase from Corynebacterium sp. U-96

Heterotetrameric sarcosine oxidase from Corynebacterium sp.U-96(SO-U96) contains non-covalent and covalent flavins. Lys-358 and Lys-171 in the beta subunit is present at non-covalent flavin adenine dinucleotide (FAD)- and covalent flavin monodinucleotide (FMN)-binding sites, respectively. The Lys-358 mutant, K358R showed 0.07% activity and higher apparent K(m) for sarcosine than the wild-type enzyme, but K358A and K358D mutants showed no activity, suggesting the importance of amino group of Lys358 in the sarcosine-binding to the enzyme. The Lys171 mutants, K171R, K171A and K171D showed 58, 39 and 32% activity of the wild-type enzyme, respectively. An apparent K(m) for oxygen and K(d) of enzyme-sulphite complex increased by the mutation. The rate of reduction of the FAD of K171 mutants with sarcosine did not change by the mutation. The stopped-flow photodiode array analyses of the anaerobic reduction with sarcosin of the wild-type and K171 mutant enzymes showed characteristic spectra of neutral and anionic semiquinones, especially for K171A enzyme. On the basis of these results, the reductive-half reaction of the wild-type and K171 mutant enzymes is explained by a mechanism involving the semiquinones. Low activity of K171 mutants is suggested to be derived from the low rate of oxidation of the reduced FMN in the enzyme.     

Quantification of allosteric influence of Escherichia coli phosphofructokinase by frequency domain fluorescence

The allosteric properties of the wild-type Escherichia coli phosphofructokinase were compared to the E187A mutant by using frequency-domain techniques. Tryptophan-shifted mutants comprising of double (W311Y/Y55W and W/311F/F188W) and triple (W311Y/Y55W/E187A and W311F/F188W/E187A) amino acid residue changes, which allowed for better fluorescence probing at targeted sites, were also compared to the wild-type and E187A. The additive nature of multiple mutations allowed one to partition the net effect of modifying residue 187. In general, the mutant enzymes displayed greater heterogeneity in sub-state population than did the wild-type enzyme. The semi-cone angle model was used to quantify the extent of depolarization of the fluorophore. Use of the model presupposes that the extent of depolarization directly correlates with the degree of flexibility of the fluorophore. A relationship has been established between the values determined from the semi-cone angle calculations and the thermodynamic components responsible for the allosteric linkage between the regulatory and substrate binding. Coupling interactions giving rise to positive entropy components are manifested by increasing flexibility of the ternary complexes rather than the binary complexes.     

Evidence for two conformational states of thioredoxin reductase from Escherichia coli: use of intrinsic and extrinsic quenchers of flavin fluorescence as probes to observe domain rotation.

Thioredoxin reductase (TrxR) from Escherichia coli consists of two globular domains connected by a two-stranded beta sheet: an FAD domain and a pyridine nucleotide binding domain. The latter domain contains the redox-active disulfide composed of Cys 135 and Cys 138. TrxR is proposed to undergo a conformational change whereby the two domains rotate 66 degrees relative to each other (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800-816), placing either redox active disulfide (FO conformation) or the NADPH binding site (FR conformation) adjacent to the flavin. This domain rotation model was investigated by using a Cys 138 Ser active-site mutant. The flavin fluorescence of this mutant is only 7% that of wild-type TrxR, presumably due to the proximity of Ser 138 to the flavin in the FO conformation. Reaction of the remaining active-site thiol, Cys 135, with phenylmercuric acetate (PMA) causes a 9.5-fold increase in fluorescence. Titration of the PMA-treated mutant with the nonreducing NADP(H) analogue, 3-aminopyridine adenine dinucleotide phosphate (AADP+), results in significant quenching of the flavin fluorescence, which demonstrates binding adjacent to the FAD, as predicted for the FR conformation. Wild-type TrxR, with or without PMA treatment, shows similar quenching by AADP+, indicating that it exists mostly in the FR conformer. These findings, along with increased EndoGluC protease susceptibility of PMA-treated enzymes, agree with the model that the FO and FR conformations are in equilibrium. PMA treatment, because of steric limitations of the phenylmercuric adduct in the FO form, forces the equilibrium to the FR conformer, where AADP+ binding can cause fluorescence quenching and conformational restriction favors proteolytic susceptibility.     

Exploring the conformational equilibrium of E. coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy

The conformational dynamics of wild-type Escherichia coli thioredoxin reductase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-resolved fluorescence of the flavin cofactor in combination with circular dichroism (both in the flavin fingerprint and far-UV regions) and steady-state fluorescence and absorption spectroscopy. The spectroscopic data show two conformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably. Ultrafast fluorescence lifetime measurements make it possible to distinguish between the two different populations: Dominant picosecond lifetimes of approximately 1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S. Long-lived fluorescence with two time constants in the range of 0.2-1 ns (total contribution 17%) originates from enzyme molecules in the FR conformation. The near absence of fast lifetime components in oxidized wild-type TrxR supports the idea of this enzyme being predominantly in the FR conformation. The emission spectrum of the FO conformation is blue-shifted with respect to that of the FR conformation. Because of the large difference in fluorescence characteristics, fluorescence measurements on time scales longer than 100 ps are fully determined by the fraction of enzyme molecules in the FR conformation. Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme and TrxR C138S stabilizes the enzymes in the FR conformation. Specific binding of the NADPH-analog, AADP(+), to the FR conformation resulted in dynamic fluorescence quenching in support of the multiple quenching sites model. Raising the temperature from 277K-323K resulted in a moderate shift to the FR conformation for TrxR C138S. High concentrations of the cosolvent glycerol triggered the domain rotation from the FO to the FR conformation.     

Time-resolved fluorescence studies of genetically engineered Escherichia coli glutamine synthetase. Effects of ATP on the tryptophan-57 loop.

Single-tryptophan-containing mutants of low adenylation state Escherichia coli glutamine synthetase (wild type has two tryptophans at positions 57 and 158) have been constructed and studied by multifrequency phase/modulation fluorescence spectroscopy. The W57L mutant (retains tryptophan at residue 158) and the W158S mutant (retains tryptophan at residue 57) are both characterized by heterogeneous exponential decay kinetics. Global analysis indicates that for the Mn-bound form of the enzyme at pH 7.4 the fluorescence of both tryptophans is best described by a sum of three discrete expontials with recovered lifetimes of 4.77, 1.72, and 0.10 ns for Trp-57 and 5.04, 2.28, and 0.13 ns for Trp-158. The wild-type enzyme also exhibits decay kinetics described by a triple-exponential model with similar lifetime components. The individual tryptophans are distinguishable by the fractional intensities of the resolvable lifetimes. The wild-type and W158S enzymes are dominated by the 5-ns component which provides nearly 60% and 65%, respectively, of the fractional intensity at five wavelengths spanning the emission spectrum. In contrast, the W57L enzyme demonstrates a larger fraction of the 2-ns lifetime species (60%) and only 35% of the longer lifetime component. The substrate ATP induces a shift to approximately 90% of the 5-ns component for the wild-type and W158S enzymes, whereas the W57L protein is essentially unaffected by this ligand. Steady-state quenching studies with iodide indicate that addition of ATP results in a 3.0-3.5-fold decrease in the apparent Stern-Volmer quenching constants for the wild-type and W158S enzymes. Phase/modulation experiments at several iodide concentrations indicate that the median, 2 ns, lifetime component is selectively quenched compared to the 5-ns lifetime component. These results suggest a model where ATP binding results in a shift in the equilibrium distribution of microconformational states populated by Trp-57. ATP shifts this equilibrium nearly completely to the states exhibiting the long-lifetime component which, based on quenching studies, is less solvent-accessible than the conformational states associated with the other lifetime components.     

Characterization of active site residues of nitroalkane oxidase

The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by ∼5-fold and decreases in the rate constant for product release of ∼2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure.     

Rapid relaxation processes in pig heart lipoamide dehydrogenase revealed by subnanosecond resolved fluorometry

The decay kinetics of the FAD-fluorescence in lipoamide dehydrogenase from pig heart have been reinvestigated using phase fluorometric methods and sophisticated laser pulse techniques. Both pulse and modulation methods lead to distinct heterogeneity in lifetimes. The two different techniques lead to good correspondence in the longer lifetime component of a biexponential decay model, whereas the more rapidly decaying component is distinctly shorter and has a larger amplitude using the phase technique with two available modulation frequencies (15 and 60 MHz). Lifetime measurements as a function of temperature and in the presence of D₂O instead of H₂O illustrate that the quenching of the FAD fluorescence in lipoamide. dehydrogenase is mainly dynamic in nature and that solvent comes into contact with the fluorophor. Mobility of the flavin itself, free and bound to the enzyme, has been measured by both differential polarized phase fluorometry and experimental fluorescence anisotropy decay after ps laser pulse excitation. By employing flavin models it has been shown that both techniques have ps time resolution. Measurements with the latter more direct method indicate a rapid subnanosecond motion of the FAD bound within the enzyme, only visible at temperatures lower than about 15°C, where the protein rotational diffusion is slowed down. The significance of rapid transient conformational fluctuations for catalysis is discussed with reference to recently developed insights reported in the literature.     

Steady state and picosecond time-resolved fluorescence studies on native, desulpho and deflavo xanthine oxidase

Steady state and time-resolved fluorescence studies on native, desulpho and deflavo xanthine oxidase (XO) have been carried out to investigate the conformational changes associated with the replacement of the molybdenum double bonded sulphur by oxygen and the removal of the flavin adenine dinucleotide (FAD). The steady state quenching experiments of the intrinsic tryptophan residues of the enzyme show that all the nine tryptophans are accessible to neutral quencher, acrylamide, in the native as well as desulpho and deflavo enzymes. However, the number of the tryptophan residues accessible to the ionic quenchers, potassium iodide and cesium chloride, increases upon removal of the FAD centre from the enzyme. This indicates that two tryptophan residues move out from the core of the enzyme to the solvent upon the removal of the FAD. The time-resolved fluorescence studies were carried out on the native, desulpho and deflavo XO by means of the time-correlated single photon counting technique, and the data were analysed by discrete exponential and maximum entropy methods. The results show that the fluorescence decay curve fitted best to a three-exponential model with lifetimes tau(1)=0.4, tau(2)=1.4 and tau(3)=3.0 ns for the native and desulpho XO, and tau(1)=0.7, tau(2)=1.7 and tau(3)=4.8 ns for the deflavo XO. The replacement of the molybdenum double bonded sulphur by oxygen in the desulpho enzyme does not cause any significant change of the lifetime components. However, removal of the FAD centre causes a significant change in the shortest and longest lifetime components indicating a conformational change in the deflavo XO possibly in the flavin domain. Decay-associated emission spectra at various emission wavelengths have been used to determine the origin of the lifetimes. The results show that tau(1) and tau(3) of the native and desulpho XO originate from the tryptophan residues which are completely or partially accessible to the solvent but tau(2) corresponds to those residues which are buried in the core of the enzyme and not exposed to the solvent. For deflavo enzyme, tau(2) is red shifted compared to the native enzyme indicating the movement of tryptophan residues from the core of the enzyme to the solvents.     

Arginine 91 is not essential for flavin incorporation in hepatic cytochrome b(5) reductase

Cytochrome b(5) reductase (cb5r) catalyzes the transfer of reducing equivalents from NADH to cytochrome b(5). Utilizing an efficient heterologous expression system that produces a histidine-tagged form of the hydrophilic, diaphorase domain of the enzyme, site-directed mutagenesis has been used to generate cb5r mutants with substitutions at position 91 in the primary sequence. Arginine 91 is an important residue in binding the FAD prosthetic group and part of a conserved "RxY(T)(S)xx(S)(N)" sequence motif that is omnipresent in the "ferredoxin:NADP(+) reductase" family of flavoproteins. Arginine 91 was replaced with K, L, A, P, D, Q, and H residues, respectively, and all the mutant proteins purified to homogeneity. Individual mutants were expressed with variable efficiency and all exhibited molecular masses of approximately 32 kDa. With the exception of R91H, all the mutants retained visible absorption spectra typical of a flavoprotein, the former being produced as an apoprotein. Visible absorption spectra of R91A, L, and P were red shifted with maxima at 458 nm, while CD spectra indicated an altered FAD environment for all the mutants except R91K. Fluorescence spectra showed a reduced degree of intrinsic flavin fluorescence quenching for the R91K, A, and P, mutants, while thermal stability studies suggested all the mutants, except R91K, were somewhat less stable than the wild-type domain. Initial-rate kinetic measurements demonstrated that the mutants exhibited decreased NADH:ferricyanide reductase activity with the R91P mutant retaining the lowest activity, corresponding to a k(cat) of 283 s(-1) and a K(NADH)(m) of 105 microM, when compared to the wild-type domain (k(cat) = 800 s(-1) K(NADH)(m) = 6 microM). These results demonstrate that R91 is not essential for FAD binding in cb5r; however, mutation of R91 perturbs the flavin environment and alters both diaphorase substrate recognition and utilization.     

Properties of lipoamide dehydrogenase altered by site-directed mutagenesis at a key residue (I184Y) in the pyridine nucleotide binding domain.

The binding of pyridine nucleotide to human erythrocyte glutathione reductase, an enzyme of known three-dimensional structure, requires some movement of the side chain of Tyr197. Moreover, this side chain lies very close to the isoalloxazine ring of the FAD cofactor. The analogous residue, Ile184, in the homologous enzyme Escherichia coli lipoamide dehydrogenase has been altered by site-directed mutagenesis to a tyrosine residue (I184Y) [Russell, G. C., Allison, N., Williams, C. H., Jr., & Guest, J.R. (1989) Ann. N.Y. Acad. Sci. 573, 429-431]. Characterization of the altered enzyme shows that the rate of the pyridine nucleotide half-reaction has been markedly reduced and that the spectral properties have been changed to mimic those of glutathione reductase. Therefore, Ile184 is shown to be an important residue in modulating the properties of the flavin in lipoamide dehydrogenase. Turnover in the dihydrolipoamide/NAD+ reaction is decreased by 10-fold and in the NADH/lipoamide reaction by 2-fold in I184Y lipoamide dehydrogenase. The oxidized form of I184Y shows remarkable changes in the fine structure of the visible absorption and circular dichroism spectra and also shows nearly complete quenching of FAD fluorescence. The spectral properties of the altered enzyme are thus similar to those of glutathione reductase and very different from those of wild-type lipoamide dehydrogenase. On the other hand, spectral evidence does not reveal any change in the amount of charge-transfer stabilization at the EH2 level. Stopped-flow data indicate that, in the reduction of I184Y by NADH, the first step, reduction of the flavin, is only slightly slowed but the subsequent two-electron transfer to the disulfide is markedly inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of FAD reduction and role of active site residues His-225 and Tyr-259 in Arthrobacter globiformis dimethylglycine oxidase: analysis of mutant structure and catalytic function

Residues His-225 and Tyr-259 are located close to the FAD in the dehydrogenase active site of the bifunctional dimethylglycine oxidase (DMGO) of Arthrobacter globiformis. We have suggested [Leys, D., Basran, J., and Scrutton, N. S. (2003) EMBO J. 22, 4038-4048] that these residues are involved in abstraction of a proton from the substrate amine group of dimethylglycine prior to C-H bond breakage and FAD reduction. To investigate this proposal, we have isolated two mutant forms of DMGO in which (i) His-225 is replaced with Gln-225 (H225Q mutant) and (ii) Tyr-259 is replaced with Phe-259 (Y259F mutant). Both mutant enzymes retain the ability to oxidize substrate, but the steady-state turnover of the Y259F mutant is attenuated more than 200-fold. Only modest changes in kinetic parameters are observed for the H225Q mutant during steady-state turnover. Stopped-flow studies indicate that the rate of FAD reduction in the Y259F enzyme is substantially impaired by a factor of approximately 1500 compared with that of the wild-type enzyme, suggesting a key role for this residue in the reductive half-reaction of the enzyme. The kinetics of FAD reduction in the H225Q enzyme are complex and involve three discrete kinetic phases that are attributed to different conformational states of this mutant, evidence for which is provided by crystallographic analysis. Neither the H225Q enzyme nor the Y259F enzyme stabilizes the FADH(2)-iminium charge-transfer complex observed previously in stopped-flow studies with the wild-type enzyme. Our studies are consistent with a key role for Tyr-259, but not His-225, in deprotonation of the substrate amine group prior to FAD reduction. We infer that residue His-225 is likely to modulate the acid-base properties of Tyr-259 by perturbing the pK(a) of Tyr-259 and thus fine-tunes the reaction chemistry to facilitate proton abstraction under physiological conditions. Our data are discussed in the context of the crystallographic data for DMGO and also in relation to contemporary mechanisms for flavoprotein-catalyzed oxidation of amine substrates.     

Identification of a glycine-rich sequence as an NAD(P)H-binding site and tyrosine 128 as a dicumarol-binding site in rat liver NAD(P)H:quinone oxidoreductase by site-directed mutagenesis.

Site-directed mutagenesis was utilized to identify binding sites for NAD(P)H and dicumarol in rat liver NAD(P)H:quinone oxidoreductase (NQOR, EC 1.6.99.2). The mutant cDNA clones were generated by a procedure based on the polymerase chain reaction and were expressed in Escherichia coli. The mutant enzymes were purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and were found to contain 2 FADs/enzyme molecule identical with that of the wild-type NQOR. Purified mutant enzymes Y128D, G150F, G150V, S151F, and Y155D showed dramatic decreases in activities in the reduction of dichlorophenolindophenol in comparison with the activities of the wild-type enzyme, whereas the activities of F124L, T127V, T127E, Y128V, Y128F, S151A, and Y155V were similar to those of NQOR. Enzyme kinetic analysis revealed that the Km values of T127E, Y128D, G150F, G150V, S151F, and Y155D were, respectively, 4-, 2-, 13-, 5-, 26-, and 19-fold higher than the Km of NQOR for NADPH, and were, respectively, 2-, 3-, 7-, 3-, 20-, and 11-fold higher than that of NQOR for NADH. The kcat values of Y128D, G150F, and G150V were also much lower than those of NQOR, but the kcat values of other mutants were similar to those of the wild-type enzyme. The Km values of the mutants for dichlorophenolindophenol were the same or slightly higher than that of NQOR. The apparent inhibition constants (Ki) for dicumarol on Y128V and F124L were elevated 12 and 8 times, respectively. Similar, but smaller, changes on Ki for 4-hydroxycoumarin were also observed. This study demonstrated that residues Gly150, Ser151, and Tyr155 in the glycine-rich region of NQOR are essential for NADPH and NADH binding and Tyr128 is important for dicumarol binding. Based on the results of the study, it is proposed that the glycine-rich region of the enzyme, along with other residues around the region, forms a beta sheet-turn-alpha helix structure important for the binding of the pyrophosphate group of NADPH and NADH.     

Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae.

The FAD1 gene of Saccharomyces cerevisiae has been selected from a genomic library on the basis of its ability to partially correct the respiratory defect of pet mutants previously assigned to complementation group G178. Mutants in this group display a reduced level of flavin adenine dinucleotide (FAD) and an increased level of flavin mononucleotide (FMN) in mitochondria. The restoration of respiratory capability by FAD1 is shown to be due to extragenic suppression. FAD1 codes for an essential yeast protein, since disruption of the gene induces a lethal phenotype. The FAD1 product has been inferred to be yeast FAD synthetase, an enzyme that adenylates FMN to FAD. This conclusion is based on the following evidence. S. cerevisiae transformed with FAD1 on a multicopy plasmid displays an increase in FAD synthetase activity. This is also true when the gene is expressed in Escherichia coli. Lastly, the FAD1 product exhibits low but significant primary sequence similarity to sulfate adenyltransferase, which catalyzes a transfer reaction analogous to that of FAD synthetase. The lower mitochondrial concentration of FAD in G178 mutants is proposed to be caused by an inefficient exchange of external FAD for internal FMN. This is supported by the absence of FAD synthetase activity in yeast mitochondria and the presence of both extramitochondrial and mitochondrial riboflavin kinase, the preceding enzyme in the biosynthetic pathway. A lesion in mitochondrial import of FAD would account for the higher concentration of mitochondrial FMN in the mutant if the transport is catalyzed by an exchange carrier. The ability of FAD1 to suppress impaired transport of FAD is explained by mislocalization of the synthetase in cells harboring multiple copies of the gene. This mechanism of suppression is supported by the presence of mitochondrial FAD synthetase activity in S. cerevisiae transformed with FAD1 on a high-copy-number plasmid but not in mitochondrial of a wild-type strain.     

Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin

The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) is oxidized to the widely used flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde). The first step of this reaction is extremely slow due to the formation of a covalent FAD N-5-creosol adduct. After a single round of error-prone PCR, seven mutants were generated with increased reactivity to creosol. The single-point mutants I238T, F454Y, E502G, and T505S showed an up to 40-fold increase in catalytic efficiency (kcat/Km) with creosol compared with the wild-type enzyme. This enhanced reactivity was due to a lower stability of the covalent flavin-substrate adduct, thereby promoting vanillin formation. The catalytic efficiencies of the mutants were also enhanced for other ortho-substituted 4-methylphenols, but not for p-cresol (4-methylphenol). The replaced amino acid residues are not located within a distance of direct interaction with the substrate, and the determined three-dimensional structures of the mutant enzymes are highly similar to that of the wild-type enzyme. These results clearly show the importance of remote residues, not readily predicted by rational design, for the substrate specificity of enzymes.