Characterization of mutants that change the hydrogen bonding of the semiquinone radical at the QH site of the cytochrome bo3 from Escherichia coli

The cytochrome bo3 ubiquinol oxidase catalyzes the two-electron oxidation of ubiquinol in the cytoplasmic membrane of Escherichia coli, and reduces O2 to water. This enzyme has a high affinity quinone binding site (QH), and the quinone bound to this site acts as a cofactor, necessary for rapid electron transfer from substrate ubiquinol, which binds at a separate site (QL), to heme b. Previous pulsed EPR studies have shown that a semiquinone at the QH site formed during the catalytic cycle is a neutral species, with two strong hydrogen bonds to Asp-75 and either Arg-71 or Gln-101. In the current work, pulsed EPR studies have been extended to two mutants at the QH site. The D75E mutation has little influence on the catalytic activity, and the pattern of hydrogen bonding is similar to the wild type. In contrast, the D75H mutant is virtually inactive. Pulsed EPR revealed significant structural changes in this mutant. The hydrogen bond to Arg-71 or Gln-101 that is present in both the wild type and D75E mutant oxidases is missing in the D75H mutant. Instead, the D75H has a single, strong hydrogen bond to a histidine, likely His-75. The D75H mutant stabilizes an anionic form of the semiquinone as a result of the altered hydrogen bond network. Either the redistribution of charge density in the semiquinone species, or the altered hydrogen bonding network is responsible for the loss of catalytic function.     

Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems

The antimycin-sensitive ubisemiquinone radical (QC) of the ubiquinol-cytochrome c oxidoreductase of submitochondrial particles and chromatophores of Rhodopseudomonas sphaeroides Ga has been studied by a combination of redox potentiometry and EPR spectroscopy. This g = 2.005 radical signal appears at physiological pH values and increases in intensity with increasing pH up to pH 7.6 in submitochondrial particles and pH 9.0 in R. sphaeroides after which its intensity remains unchanged. The Em7 (ubiquinone/quinol) of the signal, estimated from redox titration data is 80 mV for submitochondrial particles, and 150 mV in chromatophores. Each of these values is higher than that of the quinone pool by 20 mV in submitochondrial particles and 60 mV in R. sphaeroides. This indicates that the quinone at the binding site is out of equilibrium with the pool, and that binding site preferentially binds quinol over quinone. Analysis of the shapes of the semiquinone titration curves, taken together with the midpoint elevation, indicates a quinone-binding site: cytochrome c1 stoichiometry of 1:1 in both submitochondrial particles and chromatophores. At its maximal intensity, the semiquinone concentration at the binding site is 0.26 in submitochondrial particles (greater than pH 7.6) and 0.4 in chromatophores (greater than pH 9.0). In both systems, the midpoint of the ubiquinone/ubisemiquinone couple is constant as the pH is raised up to the pH of maximal semiquinone formation whereafter it becomes more negative at the rate of -60 mV/pH unit. The midpoint of the ubisemiquinone/quinol couple, on the other hand, varies by -120 mV/pH unit at pH values up to the transition pH, after which it, too, changes by -60 mV/pH unit. This seemingly anomalous behavior may be explained by invoking a protonated group at or near the quinone-binding site whose pK corresponds to the pH transition point in the quinone/semiquinone/quinol redox chemistry when the site is free or when quinone or quinol occupies the site. This pK is elevated to at least pH 9.0 in submitochondrial particles and 10.5 in R. sphaeroides when semiquinone is bound to the site.     

Retention of heme in axial ligand mutants of succinate-ubiquinone xxidoreductase (complex II) from Escherichia coli

Succinate-ubiquinone oxidoreductase (SdhCDAB, complex II) from Escherichia coli is a four-subunit membrane-bound respiratory complex that catalyzes ubiquinone reduction by succinate. In the E. coli enzyme, heme b(556) is ligated between SdhC His(84) and SdhD His(71). Contrary to a previous report (Vibat, C. R. T., Cecchini, G., Nakamura, K., Kita, K., and Gennis, R. B. (1998) Biochemistry 37, 4148-4159), we demonstrate the presence of heme in both SdhC H84L and SdhD H71Q mutants of SdhCDAB. EPR spectroscopy reveals the presence of low spin heme in the SdhC H84L (g(z) = 2.92) mutant and high spin heme in the SdhD H71Q mutant (g = 6.0). The presence of low spin heme in the SdhC H84L mutant suggests that the heme b(556) is able to pick up another ligand from the protein. CO binds to the reduced form of the mutants, indicating that it is able to displace one of the ligands to the low spin heme of the SdhC H84L mutant. The g = 2.92 signal of the SdhC H84L mutant titrates with a redox potential at pH 7.0 (E(m)(,7)) of approximately +15 mV, whereas the g = 6.0 signal of the SdhD H71Q mutant titrates with an E(m)(,7) of approximately -100 mV. The quinone site inhibitor pentachlorophenol perturbs the heme optical spectrum of the wild-type and SdhD H71Q mutant enzymes but not the SdhC H84L mutant. This finding suggests that the latter residue also plays an important role in defining the quinone binding site of the enzyme. The SdhC H84L mutation also results in a significant increase in the K(m) and a decrease in the k(cat) for ubiquinone-1, whereas the SdhD H71Q mutant has little effect on these parameters. Overall, these data indicate that SdhC His(84) has an important role in defining the interaction of SdhCDAB with both quinones and heme b(556).     

Interactions of intermediate semiquinone with surrounding protein residues at the Q(H) site of wild-type and D75H mutant cytochrome bo3 from Escherichia coli

Selective (15)N isotope labeling of the cytochrome bo(3) ubiquinol oxidase from Escherichia coli with auxotrophs was used to characterize the hyperfine couplings with the side-chain nitrogens from residues R71, H98, and Q101 and peptide nitrogens from residues R71 and H98 around the semiquinone (SQ) at the high-affinity Q(H) site. The two-dimensional ESEEM (HYSCORE) data have directly identified N(ε) of R71 as an H-bond donor carrying the largest amount of unpaired spin density. In addition, weaker hyperfine couplings with the side-chain nitrogens from all residues around the SQ were determined. These hyperfine couplings reflect a distribution of the unpaired spin density over the protein in the SQ state of the Q(H) site and the strength of interaction with different residues. The approach was extended to the virtually inactive D75H mutant, where the intermediate SQ is also stabilized. We found that N(ε) of a histidine residue, presumably H75, carries most of the unpaired spin density instead of N(ε) of R71, as in wild-type bo(3). However, the detailed characterization of the weakly coupled (15)N atoms from selective labeling of R71 and Q101 in D75H was precluded by overlap of the (15)N lines with the much stronger ~1.6 MHz line from the quadrupole triplet of the strongly coupled (14)N(ε) atom of H75. Therefore, a reverse labeling approach, in which the enzyme was uniformly labeled except for selected amino acid types, was applied to probe the contribution of R71 and Q101 to the (15)N signals. Such labeling has shown only weak coupling with all nitrogens of R71 and Q101. We utilize density functional theory-based calculations to model the available information about (1)H, (15)N, and (13)C hyperfine couplings for the Q(H) site and to describe the protein-substrate interactions in both enzymes. In particular, we identify the factors responsible for the asymmetric distribution of the unpaired spin density and ponder the significance of this asymmetry to the quinone's electron transfer function.     

High-stability semiquinone intermediate in nitrate reductase A (NarGHI) from Escherichia coli is located in a quinol oxidation site close to heme bD

Quinol/nitrate oxidoreductase (NarGHI) is the first enzyme involved in respiratory denitrification in prokaryotes. Although this complex in E. coli is known to operate with both ubi and menaquinones, the location and the number of quinol binding sites remain elusive. NarGHI strongly stabilizes a semiquinone radical located within the dihemic anchor subunit NarI. To identify its location and function, we used a combination of mutagenesis, kinetics, EPR, and ENDOR spectroscopies. For the NarGHIH66Y and NarGHIH187Y mutants lacking the distal heme bD, no EPR signal of the semiquinone was observed. In contrast, a semiquinone was detected in the NarGHIH56Y mutant lacking the proximal heme bP. Its thermodynamic properties and spectroscopic characteristics, as revealed by Q-band EPR and ENDOR spectroscopies, are identical to those observed in the native enzyme. The substitution by Ala of the Lys86 residue close to heme bD, which was previously proposed to be in a quinol oxidation site of NarGHI (QD), also leads to the loss of the EPR signal of the semiquinone, although both hemes are present. Enzymatic assays carried out on the NarGHIK86A mutant reveal that the substitution dramatically reduces the rate of oxidation of both mena and ubiquinol analogues. These observations demonstrate that the semiquinone observed in NarI is strongly associated with heme bD and that Lys86 is required for its stabilization. Overall, our results indicate that the semiquinone is located within the quinol oxidation site QD. Details of the possible binding motif of the semiquinone and mechanistic implications are discussed.     

Crystallographic investigation of the role of aspartate 95 in the modulation of the redox potentials of Desulfovibrio vulgaris flavodoxin

The side chain of aspartate 95 in flavodoxin from Desulfovibrio vulgaris provides the closest negative charge to N(1) of the bound FMN in the protein. Site-directed mutagenesis was used to substitute alanine, asparagine, or glutamate for this amino acid to assess the effect of this charge on the semiquinone/hydroquinone redox potential (E(1)) of the FMN cofactor. The D95A mutation shifts the E(1) redox potential positively by 16 mV, while a negative shift of 23 mV occurs in the oxidized/semiquinone midpoint redox potential (E(2)). The crystal structures of the oxidized and semiquinone forms of this mutant are similar to the corresponding states of the wild-type protein. In contrast to the wild-type protein, a further change in structure occurs in the D95A mutant in the hydroquinone form. The side chain of Y98 flips into an energetically more favorable edge-to-face interaction with the bound FMN. Analysis of the structural changes in the D95A mutant, taking into account electrostatic interactions at the FMN binding site, suggests that the pi-pi electrostatic repulsions have only a minor contribution to the very low E(1) redox potential of the FMN cofactor when bound to apoflavodoxin. Substitution of D95 with glutamate causes only a slight perturbation of the two one-electron redox potentials of the FMN cofactor. The structure of the D95E mutant reveals a large movement of the 60-loop (residues 60-64) away from the flavin in the oxidized structure. Reduction of this mutant to the hydroquinone causes the conformation of the 60-loop to revert back to that occurring in the structures of the wild-type protein. The crystal structures of the D95E mutant imply that electrostatic repulsion between a carboxylate on the side chain at position 95 and the phenol ring of Y98 prevents rotation of the Y98 side chain to a more energetically favorable conformation as occurs in the D95A mutant. Replacement of D95 with asparagine has no effect on E(2) but causes E(1) to change by 45 mV. The D95N mutant failed to crystallize. The K(d) values of the protein FMN complex in all three oxidation-reduction states differ from those of the wild-type complexes. Molecular modeling showed that the conformational energy of the protein changes with the redox state, in qualitative agreement with the observed changes in K(d), and allowed the electrostatic interactions between the FMN and the surrounding groups on the protein to be quantified.     

Covalent flavinylation is essential for efficient redox catalysis in vanillyl-alcohol oxidase

By mutating the target residue of covalent flavinylation in vanillyl-alcohol oxidase, the functional role of the histidyl-FAD bond was studied. Three His(422) mutants (H422A, H422T, and H422C) were purified, which all contained tightly but noncovalently bound FAD. Steady state kinetics revealed that the mutants have retained enzyme activity, although the turnover rates have decreased by 1 order of magnitude. Stopped-flow analysis showed that the H422A mutant is still able to form a stable binary complex of reduced enzyme and a quinone methide product intermediate, a crucial step during vanillyl-alcohol oxidase-mediated catalysis. The only significant change in the catalytic cycle of the H422A mutant is a marked decrease in reduction rate. Redox potentials of both wild type and H422A vanillyl-alcohol oxidase have been determined. During reduction of H422A, a large portion of the neutral flavin semiquinone is observed. Using suitable reference dyes, the redox potentials for the two one-electron couples have been determined: -17 and -113 mV. Reduction of wild type enzyme did not result in any formation of flavin semiquinone and revealed a remarkably high redox potential of +55 mV. The marked decrease in redox potential caused by the missing covalent histidyl-FAD bond is reflected in the reduced rate of substrate-mediated flavin reduction limiting the turnover rate. Elucidation of the crystal structure of the H422A mutant established that deletion of the histidyl-FAD bond did not result in any significant structural changes. These results clearly indicate that covalent interaction of the isoalloxazine ring with the protein moiety can markedly increase the redox potential of the flavin cofactor, thereby facilitating redox catalysis. Thus, formation of a histidyl-FAD bond in specific flavoenzymes might have evolved as a way to contribute to the enhancement of their oxidative power.     

Cholesterol oxidase from Brevibacterium sterolicum. The relationship between covalent flavinylation and redox properties

Brevibacterium sterolicum possesses two forms of cholesterol oxidase, one containing noncovalently bound FAD, the second containing a FAD covalently linked to His(69) of the protein backbone. The functional role of the histidyl-FAD bond in the latter cholesterol oxidase was addressed by studying the properties of the H69A mutant in which the FAD is bound tightly, but not covalently, and by comparison with native enzyme. The mutant retains catalytic activity, but with a turnover rate decreased 35-fold; the isomerization step of the intermediate 3-ketosteroid to the final product is also preserved. Stabilization of the flavin semiquinone and binding of sulfite are markedly decreased, this correlates with a lower midpoint redox potential (-204 mV compared with -101 mV for wild-type). Reconstitution with 8-chloro-FAD led to a holoenzyme form of H69A cholesterol oxidase with a midpoint redox potential of -160 mV. In this enzyme form, flavin semiquinone is newly stabilized, and a 3.5-fold activity increase is observed, this mimicking the thermodynamic effects induced by the covalent flavin linkage. It is concluded that the flavin 8alpha-linkage to a (N1)histidine is a pivotal factor in the modulation of the redox properties of this cholesterol oxidase to increase its oxidative power.     

The role of double covalent flavin binding in chito-oligosaccharide oxidase from Fusarium graminearum

ChitO (chito-oligosaccharide oxidase) from Fusarium graminearum catalyses the regioselective oxidation of N-acetylated oligosaccharides. The enzyme harbours an FAD cofactor that is covalently attached to His94 and Cys154. The functional role of this unusual bi-covalent flavin-protein linkage was studied by site-directed mutagenesis. The double mutant (H94A/C154A) was not expressed, which suggests that a covalent flavin-protein bond is needed for protein stability. The single mutants H94A and C154A were expressed as FAD-containing enzymes in which one of the covalent FAD-protein bonds was disrupted relative to the wild-type enzyme. Both mutants were poorly active, as the k(cat) decreased (8.3- and 3-fold respectively) and the K(m) increased drastically (34- and 75-fold respectively) when using GlcNac as the substrate. Pre-steady-state analysis revealed that the rate of reduction in the mutant enzymes is decreased by 3 orders of magnitude when compared with wild-type ChitO (k(red)=750 s(-1)) and thereby limits the turnover rate. Spectroelectrochemical titrations revealed that wild-type ChitO exhibits a relatively high redox potential (+131 mV) and the C154A mutant displays a lower potential (+70 mV), while the H94A mutant displays a relatively high potential of approximately +164 mV. The results show that a high redox potential is not the only prerequisite to ensure efficient catalysis and that removal of either of the covalent bonds may perturb the geometry of the Michaelis complex. Besides tuning the redox properties, the bi-covalent binding of the FAD cofactor in ChitO is essential for a catalytically competent conformation of the active site.     

Redox potentials of flavocytochromes c from the phototrophic bacteria, Chromatium vinosum and Chlorobium thiosulfatophilum.

The redox potentials of flavocytochromes c (FC) from Chromatium vinosum and Chlorobium thiosulfatophilum have been studied as a function of pH. Chlorobium FC has a single heme which has a redox potential of +98 mV at pH 7 (N = 1) that is independent of pH between 6 and 8. The average two-electron redox potential of the flavin extrapolated to pH 7 is +28 mV and decreases 35 mV/pH between pH 6 and 7. The anionic form of the flavin semiquinone is stabilized above pH 6. The redox potential of Chromatium FC is markedly lower than for Chlorobium. The two hemes in Chromatium FC appear to have a redox potential of 15 mV at pH 7 (N = 1), although they reside in very different structural environments. The hemes of Chromatium FC have a pH-dependent redox potential, which can be fit in the simplest case by a single ionization with pK = 7.05. The flavin in Chromatium FC has an average two-electron redox potential of -26 mV at pH 7 and decreases 30 mV/pH between pH 6 and 8. As with Chlorobium, the anionic form of the flavin semiquinone of Chromatium FC is stabilized above pH 6. The unusually high redox potential of the flavin, a stabilized anion radical, and sulfite binding to the flavin in both Chlorobium and Chromatium FCs are characteristics shared by the flavoprotein oxidases. By analogy with glycolate oxidase and lactate dehydrogenase for which there are three-dimensional structures, the properties of the FCs are likely to be due to a positively charged amino acid side chain in the vicinity of the N1 nitrogen of the flavin.     

Evidence for an EPR-detectable semiquinone intermediate stabilized in the membrane-bound subunit NarI of nitrate reductase A (NarGHI) from Escherichia coli

Nitrate reductase A (NRA, NarGHI) is expressed in Escherichia coli by growing the bacterium in anaerobic conditions in the presence of nitrate. This enzyme reduces nitrate to nitrite and uses menaquinol (or ubiquinol) as the electron donor. The location of quinones in the enzyme, their number, and their role in the electron transfer mechanism are still controversial. In this work, we have investigated the spectroscopic and thermodynamic properties of a semiquinone (SQ) in membrane samples of overexpressed E. coli nitrate reductase poised in appropriate redox conditions. This semiquinone is highly stabilized with respect to free semiquinone. The g-values determined from the numerical simulation of its Q-band (35 GHz) EPR spectrum are equal to 2.0061, 2.0051, 2.0023. The midpoint potential of the Q/QH(2) couple is about -100 mV, and the SQ stability constant is about 100 at pH 7.5. The semiquinone EPR signal disappears completely upon addition of the quinol binding site inhibitor 2-n-nonyl-4-hydroxyquinoline N-oxide (NQNO). A semiquinone radical could also be stabilized in preparations where only the NarI membrane subunit is overexpressed in the absence of the NarGH catalytic dimer. Its thermodynamic and spectroscopic properties show only slight variations with those of the wild-type enzyme. The X-band continuous wave (cw) electron nuclear double resonance (ENDOR) spectra of the radicals display similar proton hyperfine coupling patterns in NarGHI and in NarI, showing that they arise from the same semiquinone species bound to a single site located in the NarI membrane subunit. These results are discussed with regard to the location and the potential function of quinones in the enzyme.     

Exploring by pulsed EPR the electronic structure of ubisemiquinone bound at the QH site of cytochrome bo3 from Escherichia coli with in vivo 13C-labeled methyl and methoxy substituents

The cytochrome bo(3) ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O(2) to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. The semiquinone is also formed in the D75E mutant, where the mutation has little influence on the catalytic activity, and in the D75H mutant, which is virtually inactive. In this work, wild-type cytochrome bo(3) as well as the D75E and D75H mutant proteins were prepared with ubiquinone-8 (13)C-labeled selectively at the methyl and two methoxy groups. This was accomplished by expressing the proteins in a methionine auxotroph in the presence of l-methionine with the side chain methyl group (13)C-labeled. The (13)C-labeled quinone isolated from cytochrome bo(3) was also used for the generation of model anion radicals in alcohol. Two-dimensional pulsed EPR and ENDOR were used for the study of the (13)C methyl and methoxy hyperfine couplings in the semiquinone generated in the three proteins indicated above and in the model system. The data were used to characterize the transferred unpaired spin densities on the methyl and methoxy substituents and the conformations of the methoxy groups. In the wild type and D75E mutant, the constraints on the configurations of the methoxy side chains are similar, but the D75H mutant appears to have altered methoxy configurations, which could be related to the perturbed electron distribution in the semiquinone and the loss of enzymatic activity.     

Amino acid residues interacting with both the bound quinone and coenzyme, pyrroloquinoline quinone, in Escherichia coli membrane-bound glucose dehydrogenase

The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) as the primary component of the respiratory chain possesses a tightly bound ubiquinone (UQ) flanking pyrroloquinoline quinone (PQQ) as a coenzyme. Several mutants for Asp-354, Asp-466, and Lys-493, located close to PQQ, that were constructed by site-specific mutagenesis were characterized by enzymatic, pulse radiolysis, and EPR analyses. These mutants retained almost no dehydrogenase activity or ability of PQQ reduction. CD and high pressure liquid chromatography analyses revealed that K493A, D466N, and D466E mutants showed no significant difference in molecular structure from that of the wild-type mGDH but showed remarkably reduced content of bound UQ. A radiolytically generated hydrated electron (e(aq)(-)) reacted with the bound UQ of the wild enzyme and K493R mutant to form a UQ neutral semiquinone with an absorption maximum at 420 nm. Subsequently, intramolecular electron transfer from the bound UQ semiquinone to PQQ occurred. In K493R, the rate of UQ to PQQ electron transfer is about 4-fold slower than that of the wild enzyme. With D354N and D466N mutants, on the other hand, transient species with an absorption maximum at 440 nm, a characteristic of the formation of a UQ anion radical, appeared in the reaction of e(aq)(-), although the subsequent intramolecular electron transfer was hardly affected. This indicates that D354N and D466N are prevented from protonation of the UQ semiquinone radical. Moreover, EPR spectra showed that mutations on Asp-466 or Lys-493 residues changed the semiquinone state of bound UQ. Taken together, we reported here for the first time the existence of a semiquinone radical of bound UQ in purified mGDH and the difference in protonation of ubisemiquinone radical because of mutations in two different amino acid residues, located around PQQ. Furthermore, based on the present results and the spatial arrangement around PQQ, Asp-466 and Lys-493 are suggested to interact both with the bound UQ and PQQ in mGDH.     

Electron spin resonance studies on a flavoprotein in neutrophil plasma membranes. Redox potentials of the flavin and its participation in NADPH oxidase

Plasma membrane fractions of stimulated and resting cells were isolated from pig blood neutrophils. The midpoint redox potential (Em) of the membrane-bound flavin was determined potentiometrically by analysis of the flavin free-radical signal by electron spin resonance (ESR) spectroscopy. In both stimulated and resting cells, a peak position of the titration curve gave an Em value of -280 mV at pH 7.0 (Em7). The flavin free radical showed an ESR spectrum at g = 2.004 with a peak to peak width of 19 G, which indicates that the redox intermediate is a neutral semiquinone. Redox titrations were anaerobically examined at 25 degrees C with NADPH in place of dithionite. Addition of NADPH to plasma membranes of stimulated cells resulted in a rapid change in potential, accompanied by the formation of the ESR signal of flavin free radical. Computer simulation of the titration points gave an ambient midpoint potential of -280 mV (Em7). In contrast, those of resting cells showed a very slow change in potential and no g = 2.00 signal formation. Power saturation behavior of the ESR signal showed a marked difference between those of stimulated and resting cells. ESR characteristics of the flavin are discussed in relation to the membrane-bound NADPH oxidase.     

Comparisons of wild-type and mutant flavodoxins from Anacystis nidulans. Structural determinants of the redox potentials

The long-chain flavodoxins, with 169-176 residues, display oxidation-reduction potentials at pH 7 that vary from -50 to -260 mV for the oxidized/semiquinone (ox/sq) equilibrium and are -400 mV or lower for the semiquinone/hydroquinone (sq/hq) equilibrium. To examine the effects of protein interactions and conformation changes on FMN potentials in the long-chain flavodoxin from Anacystis nidulans (Synechococcus PCC 7942), we have determined crystal structures for the semiquinone and hydroquinone forms of the wild-type protein and for the mutant Asn58Gly, and have measured redox potentials and FMN association constants. A peptide near the flavin ring, Asn58-Val59, reorients when the FMN is reduced to the semiquinone form and adopts a conformation ("O-up") in which O 58 hydrogen bonds to the flavin N(5)H; this rearrangement is analogous to changes observed in the flavodoxins from Clostridium beijerinckii and Desulfovibrio vulgaris. On further reduction to the hydroquinone state, the Asn58-Val59 peptide in crystalline wild-type A. nidulans flavodoxin rotates away from the flavin to the "O-down" position characteristic of the oxidized structure. This reversion to the conformation found in the oxidized state is unusual and has not been observed in other flavodoxins. The Asn58Gly mutation, at the site which undergoes conformation changes when FMN is reduced, was expected to stabilize the O-up conformation found in the semiquinone oxidation state. This mutation raises the ox/sq potential by 46 mV to -175 mV and lowers the sq/hq potential by 26 mV to -468 mV. In the hydroquinone form of the Asn58Gly mutant the C-O 58 remains up and hydrogen bonded to N(5)H, as in the fully reduced flavodoxins from C. beijerinckii and D. vulgaris. The redox and structural properties of A. nidulans flavodoxin and the Asn58Gly mutant confirm the importance of interactions made by N(5) or N(5)H in determining potentials, and are consistent with earlier conclusions that conformational energies contribute to the observed potentials.The mutations Asp90Asn and Asp100Asn were designed to probe the effects of electrostatic interactions on the potentials of protein-bound flavin. Replacement of acidic by neutral residues at positions 90 and 100 does not perturb the structure, but has a substantial effect on the sq/hq equilibrium. This potential is increased by 25-41 mV, showing that electrostatic interaction between acidic residues and the flavin decreases the potential for conversion of the neutral semiquinone to the anionic hydroquinone. The potentials and the effects of mutations in A. nidulans flavodoxin are rationalized using a thermodynamic scheme developed for C. beijerinckii flavodoxin.     

Chemical reduction of the diferric/radical center in protein R2 from mouse ribonucleotide reductase is independent of the proposed radical transfer pathway

The rates of reduction of the diferric/radical center in mouse ribonucleotide reductase protein R2 were studied by light absorption and EPR in the native protein and in three point mutants of conserved residues involved in the proposed radical transfer pathway (D266A, W103Y) or in the unstructured C terminal domain (Y370W). The pseudo-first order rate constants for chemical reduction of the tyrosyl radical and diferric center by hydroxyurea, sodium dithionite or the dihydro form of flavin adenine dinucleotide, were comparable with or higher (particularly D266A, by dithionite) than in native R2. Molecular modeling of the D266A mutant showed that the iron/radical site should be more accessible for external reductants in the mutant than in native R2. The results indicate that no specific pathway is required for the reduction. The dihydro form of flavin adenine dinucleotide was found to be a very efficient reductant in the studied proteins compared to dithionite alone. The EPR spectra of the mixed-valent Fe(II)Fe(III) sites formed by chemical reduction in the D266A and W103Y mutants were clearly different from the spectrum observed in the native protein, indicating that the structure of the diferric site was affected by the mutations, as also suggested by the modeling study. No difference was observed between the mixed-valent EPR spectra generated by chemical reduction in Y370W mutant and native mouse R2 protein.     

Functional roles of the 6-S-cysteinyl, 8alpha-N1-histidyl FAD in glucooligosaccharide oxidase from Acremonium strictum

The crystal structure of glucooligosaccharide oxidase from Acremonium strictum was demonstrated to contain a bicovalent flavinylation, with the 6- and 8alpha-positions of the flavin isoalloxazine ring cross-linked to Cys(130) and His(70), respectively. The H70A and C130A single mutants still retain the covalent FAD, indicating that flavinylation at these two residues is independent. Both mutants exhibit a decreased midpoint potential of approximately +69 and +61 mV, respectively, compared with +126 mV for the wild type, and possess lower activities with k(cat) values reduced to approximately 2 and 5%, and the flavin reduction rate reduced to 0.6 and 14%. This indicates that both covalent linkages increase the flavin redox potential and alter the redox properties to promote catalytic efficiency. In addition, the isolated H70A/C130A double mutant does not contain FAD, and addition of exogenous FAD was not able to restore any detectable activity. This demonstrates that the covalent attachment is essential for the binding of the oxidized cofactor. Furthermore, the crystal structure of the C130A mutant displays conformational changes in several cofactor and substrate-interacting residues and hence provides direct evidence for novel functions of flavinylation in assistance of cofactor and substrate binding. Finally, the wild-type enzyme is more heat and guanidine HCl-resistant than the mutants. Therefore, the bicovalent flavin linkage not only tunes the redox potential and contributes to cofactor and substrate binding but also increases structural stability.     

Identification of a signal transduction switch in the chemokine receptor CXCR1

Chemokine receptors belong to the superfamily of G protein-coupled receptors, which regulate the trafficking and activation of leukocytes, and operate as coreceptors in the entry of HIV-1. To investigate the early steps in the signal transmission from the chemokine-binding site to the G protein-coupling region we engineered metal ion-binding sites at putative extracellular sites in the chemokine receptor CXCR1. We introduced histidines into sites located in the second and third putative extracellular loops of CXCR1, creating single, double, and triple mutant receptors: R199H, R203H, D265H, R199H/R203H, R199H/D265H, R203H/D265H, R203H/H207Q, and R199H/R203H/D265H. Cells expressing the double mutants R199H/D265H and R203H/D265H and the triple mutant R199H/R203H/D265H failed to trigger interleukin 8-dependent calcium responses. Interestingly, calcium responses mediated by the single mutant R203H and the double mutants R199H/R203H and R203H/H207Q were blocked by Zn(II), indicating the creation of a functional metal ion-binding site. On the other hand, cells expressing all single, double, or triple histidine-substituted CXCR1 demonstrated high affinity binding to interleukin 8 in the presence and absence of metal ions. These findings indicate that occupation of the engineered metal-binding site uncouples the chemokine-binding site from the activation mechanism in CXCR1. Most importantly, we identify for the first time elements of an early signal transduction switch of chemokine receptors before the activation of cytoplasmic G proteins.     

Characterization of the exchangeable protons in the immediate vicinity of the semiquinone radical at the QH site of the cytochrome bo3 from Escherichia coli

The cytochrome bo3 ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O2 to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. Two-dimensional electron spin echo envelope modulation has been applied to explore the exchangeable protons involved in hydrogen bonding to the semiquinone by substitution of 1H2O by 2H2O. Three exchangeable protons possessing different isotropic and anisotropic hyperfine couplings were identified. The strength of the hyperfine interaction with one proton suggests a significant covalent O-H binding of carbonyl oxygen O1 that is a characteristic of a neutral radical, an assignment that is also supported by the unusually large hyperfine coupling to the methyl protons. The second proton with a large anisotropic coupling also forms a strong hydrogen bond with a carbonyl oxygen. This second hydrogen bond, which has a significant out-of-plane character, is from an NH2 or NH nitrogen, probably from an arginine (Arg-71) known to be in the quinone binding site. Assignment of the third exchangeable proton with smaller anisotropic coupling is more ambiguous, but it is clearly not involved in a direct hydrogen bond with either of the carbonyl oxygens. The results support a model that the semiquinone is bound to the protein in a very asymmetric manner by two strong hydrogen bonds from Asp-75 and Arg-71 to the O1 carbonyl, while the O4 carbonyl is not hydrogen-bonded to the protein.     

Electron paramagnetic resonance characterization of tyrosine radical, M+, in site-directed mutants of photosystem II(t).

Photosystem II contains two well-characterized tyrosine radicals, D(.) and Z(.). Z is an electron carrier between the primary chlorophyll donor and the manganese catalytic site and is essential for enzymatic function. On the other hand, D forms a stable radical with no known role in oxygen evolution. D(.) and Z(.) give rise to similar, but not identical, room temperature electron paramagnetic resonance (EPR) signals, which can be distinguished by their decay kinetics. A third room temperature EPR signal has also been observed in site-directed mutants in which a nonredox active amino acid is substituted at the D or Z site. This four-line EPR signal has been shown to have a tyrosine origin by isotopic labeling (Boerner and Barry, 1994, J. Biol. Chem. 269:134-137), but such an EPR signal has never before been observed from a tyrosyl radical. The radical giving rise to this third unique signal has been named M+. Here we provide kinetic evidence that this signal arises from a third redox active tyrosine, distinct from tyrosine D and Z, in the photosystem II reaction center. Isotopic labeling and EPR spectroscopy provide evidence that M is a covalently modified tyrosine.