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.     

Role of glutamate-59 hydrogen bonded to N(3)H of the flavin mononucleotide cofactor in the modulation of the redox potentials of the Clostridium beijerinckii flavodoxin. Glutamate-59 is not responsible for the pH dependency but contributes to the stabilization of the flavin semiquinone.

The midpoint potentials for both redox couples of the noncovalently bound flavin mononucleotide (FMN) cofactor in the flavodoxin are known to be pH dependent. While the pH dependency for the oxidized-semiquinone (ox/sq) couple is consistent with the formation of the blue neutral form of the flavin semiquinone, that of the semiquinone-hydroquinone (sq/hq) couple is more enigmatic. The apparent pK(a) of 6.7 for this couple in the flavodoxin from Clostridium beijerinckii has been attributed to the ionization of the FMN(HQ); however, nuclear magnetic resonance data strongly suggest the FMN(HQ) remains anionic over the entire pH range testable. As an alternative explanation, a specific glutamate residue (Glu59 in this flavodoxin), which is hydrogen-bonded to N(3)H of the FMN, has been postulated to be the primary redox-linked proton acceptor responsible for the pH effect in some flavodoxins. This model was directly tested in this study by permanently neutralizing Glu59 by its replacement with glutamine. This conservative substitution resulted in an increase of 86 mV (at pH 7) in midpoint potential of the sq/hq couple; however, the pH dependency of this couple was not altered. Thus, the redox-linked protonation of Glu59 clearly cannot be responsible for this effect as proposed. The pH dependency of the ox/sq couple was also similar to wild type, but the midpoint potential has decreased by 65 mV (pH 7). The K(d) values for the oxidized, semiquinone, and hydroquinone complexes increased by 43-, 590-, and 20-fold, respectively, relative to the wild type. Thus, the Glu59 to glutamine substitution substantially effects the stability of the semiquinone but, on a relative basis, slightly favors the formation of the hydroquinone. On the basis of (1)H-(15)N HSQC nuclear magnetic resonance spectroscopic studies, the increased temperature coefficients for the protons on N(3) and N(5) of the reduced FMN in E59Q suggest that the hydrogen-bonding interactions at these positions are significantly weakened in this mutant. The increase for N(5)H correlates with the reduced stability of the FMN(SQ) and the more negative midpoint potential for the ox/sq couple. On the basis of the X-ray structure, an "anchoring" role is proposed for the side chain carboxylate of Glu59 that stabilizes the structure of the 50's loop in such a way so as to promote the crucial hydrogen-bonding interaction that stabilizes the flavin semiquinone, contributing to the low potential of this flavodoxin.     

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.     

Conformational energetics of a reverse turn in the Clostridium beijerinckii flavodoxin is directly coupled to the modulation of its oxidation-reduction potentials

A surface loop in the flavodoxin from Clostridium beijerinckii comprised of residues -Met(56)-Gly-Asp-Glu(59)- forms a four-residue reverse turn which undergoes a conversion from a mix of cis/trans peptide configurations that approximate a type II configuration in the oxidized state to a type II' turn upon reduction of the bound flavin mononucleotide (FMN) cofactor. This change results in the formation of a new hydrogen bond between the N(5)H of the reduced cofactor and the carbonyl group of Gly57 of the central peptide bond of the turn, an interaction that is thought to contribute to the modulation of the oxidation-reduction potentials of the cofactor [Ludwig, M. L., Pattridge, K. A., Metzger, A. L., Dixon, M. M., Eren, M., Feng, Y., and Swenson, R. P. (1997) Biochemistry 36, 1259-1280]. In this study, the direct linkage of the conformational energetics of this turn to the stabilization of the FMN semiquinone was established by systematically replacing the second and third residues of the turn (Gly57 and Asp58) with the -Gly-Gly-, -Gly-Ala-, -Ala-Gly-, and -Ala-Ala- dipeptidyl sequences. On the basis of published position specific preferences for residues with side chains (mimicked by Ala) and glycine, a strong correlation was observed between E(ox/sq) and the calculated free-energy differences between the type II and type II' conformations of each of these sequence combinations. The -Ala-Gly- sequence, which favors the type II turn configuration primarily adopted in the oxidized state, displays a E(ox/sq) value that is about 150 mV more negative than that for the wild-type-like -Gly-Ala- sequence, which prefers the type II' conformation observed in the reduced states. The -Gly-Gly- and -Ala-Ala- mutants exhibit intermediate E(ox/sq) values consistent with their ambivalent turn preferences. The potential changes are primarily the result of alterations in the stability of the semiquinone state. These results provide more conclusive evidence for the crucial role of this conformational change in the modulation of the redox potentials of this flavodoxin. Furthermore, this study establishes a direct association between the conformational energetics of the protein, induced in this case by the sequence specificity of a beta-turn, and the differential thermodynamic stabilization of specific redox states of the cofactor, demonstrating another means by which flavoproteins can modulate the redox potentials of the bound cofactor.     

Role of hydrogen bonding interactions to N(3)H of the flavin mononucleotide cofactor in the modulation of the redox potentials of the Clostridium beijerinckii flavodoxin

The role of the hydrogen bonding interaction with the N(3)H of the flavin cofactor in the modulation of the redox properties of flavoproteins has not been extensively investigated. In the flavodoxin from Clostridium beijerinckii, the gamma-carboxylate group of glutamate-59 serves as a dual hydrogen bond acceptor with the N(3)H of flavin mononucleotide (FMN) cofactor and the amide hydrogen of the adjacent polypeptide backbone in all three oxidation states. This "bridging" interaction serves to anchor the FMN in the binding site, which, based on the E59Q mutant, indirectly affects the stability of the neutral flavin semiquinone by facilitating a strong and critical interaction at the FMN N(5)H [Bradley, L. H., and Swenson, R. P. (1999) Biochemistry 38, 12377-12386]. In this study, the specific role of the N(3)H interaction itself was investigated through the systematic replacement of Glu59 by aspartate, asparagine, and alanine in an effort to weaken, disrupt, and/or eliminate this interaction, respectively. Just as for the E59Q mutant, each replacement significantly weakened the binding of the cofactor, particularly for the semiquinone state, affecting the midpoint potentials of each one-electron couple in opposite directions. (1)H-(15)N HSQC nuclear magnetic resonance (NMR) spectroscopic studies revealed that not only was the N(3)H interaction weakened as anticipated, but so also was the hydrogen bonding interaction with the N(5)H. Using the temperature coefficients of the N(5)H to quantify and correct for changes in this interaction, the contribution of the N(3)H hydrogen bond to the binding of each redox state of the FMN was isolated and estimated. Based on this analysis, the N(3)H hydrogen bonding interaction appears to contribute primarily to the stability of the oxidized state (by as much as 2 kcal/mol) and to a lesser extent the reduced states. It is concluded that this interaction contributes only modestly (<45 mV) to the modulation of the midpoint potential for each redox couple in the flavodoxin. These conclusions are generally consistent with ab initio calculations and model studies on the non-protein-bound cofactor.     

The midpoint potentials for the oxidized-semiquinone couple for Gly57 mutants of the Clostridium beijerinckii flavodoxin correlate with changes in the hydrogen-bonding interaction with the proton on N(5) of the reduced flavin mononucleotide cofactor as measured by NMR chemical shift temperature dependencies.

In the Clostridium beijerinckii flavodoxin, the reduction of the flavin mononucleotide (FMN) cofactor is accompanied by a local conformation change in which the Gly57-Asp58 peptide bond "flips" from primarily the unusual cis O-down conformation in the oxidized state to the trans O-up conformation such that a new hydrogen bond can be formed between the carbonyl group of Gly57 and the proton on N(5) of the neutral FMN semiquinone radical [Ludwig, M. L., Pattridge, K. A., Metzger, A. L., Dixon, M. M., Eren, M., Feng, Y., and Swenson, R. P. (1997) Biochemistry 36, 1259-1280]. This interaction is thought to contribute to the relative stabilization of the flavin semiquinone and may be at least partially responsible for the substantial separation of the midpoint potentials of the two one-electron reduction steps. Through a series of amino acid substitutions, the above cited study demonstrated the critical role of the often conserved glycine residue in this process. However, it has not been directly established experimentally as to whether these substitutions brought about the changes in the midpoint potentials by altering the strength of this hydrogen-bonding interaction as proposed. In this study, the relative strengths of the FMN N(5)H.O57 hydrogen bond in wild type and the G57A, G57N, and G57T mutants were evaluated by measuring the temperature dependency of the chemical shift for the proton on N(5) of the fully reduced cofactor by 1H-15N HSQC nuclear magnetic resonance spectroscopy. Based on the established correlation between the temperature coefficient of amide protons and the strength of hydrogen bonding in small peptides, the apparent strength of the N(5)H.O57 interaction was found to depend on the properties of the side chain at position 57. The glycine residue found in the wild-type flavodoxin appears to provide the strongest interaction while the beta-branched side chain in the G57T mutant provides the weakest. A good correlation was noted between the temperature coefficients of N(5)H and the one-electron reduction potential for the ox/sq couple as well as the binding free energy of the FMN semiquinone in this group of mutants. These results provide more direct quantitative evidence that support the previous hypothesis that this conformation change and the associated formation of the hydrogen bonding interaction with N(5)H of the reduced FMN represent an important means of stabilizing the neutral semiquinone and in modulating the oxidation-reduction potentials of the flavin cofactor in this and perhaps other flavodoxins.     

A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: structural determinants of redox potential

Flavodoxin II from Azotobacter vinelandii is a "long-chain" flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three-dimensional structure determined to a resolution of 2.25 A by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five-stranded parallel beta-sheet flanked on either side by alpha-helices. An eight-residue insertion, compared with other long-chain flavodoxins, forms a short 3(10) helix preceding the start of the alpha3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent-accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E2 value in particular.     

Evaluation of the hydrogen bonding interactions and their effects on the oxidation-reduction potentials for the riboflavin complex of the Desulfovibrio vulgaris flavodoxin

The oxidation-reduction potentials for the riboflavin complex of the Desulfovibrio vulgaris flavodoxin are substantially different from those of the flavin mononucleotide (FMN) containing native protein, with the midpoint potential for the semiquinone-hydroquinone couple for the riboflavin complex being 180 mV less negative. This increase has been attributed to the absence in the riboflavin complex of unfavorable electrostatic effects of the dianionic 5'-phosphate of the FMN on the stability of the flavin hydroquinone anion. In this study, 15N and 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopic studies demonstrate that when bound to the flavodoxin, (1) the N1 of the riboflavin hydroquinone remains anionic at pH 7.0 so the protonation of the hydroquinone is not responsible for this increase, (2) the N5 position is much more exposed and may be hydrogen bonded to solvent, and (3) that while the hydrogen bonding interaction at the N3H appears stronger, that at the N5H in the reduced riboflavin is substantially weaker than for the native FMN complex. Thus, the higher reduction potential of the riboflavin complex is primarily the consequence of altered interactions with the flavin ring that affect hydrogen bonding with the N5H that disproportionately destabilize the semiquinone state of the riboflavin rather than through the absence of the electrostatic effects of the 5'-phosphate on the hydroquinone state.     

High‐resolution crystal structures reveal a mixture of conformers of the Gly61‐Asp62 peptide bond in an oxidized flavodoxin from Bacillus cereus

Flavodoxins (Flds) are small proteins that shuttle electrons in a range of reactions in microorganisms. Flds contain a redox‐active cofactor, a flavin mononucleotide (FMN), and it is well established that when Flds are reduced by one electron, a peptide bond close to the FMN isoalloxazine ring flips to form a new hydrogen bond with the FMN N5H, stabilizing the one‐electron reduced state. Here, we present high‐resolution crystal structures of Flavodoxin 1 from Bacillus cereus in both the oxidized (ox) and one‐electron reduced (semiquinone, sq) state. We observe a mixture of conformers in the oxidized state; a 50:50 distribution between the established oxidized conformation where the peptide bond is pointing away from the flavin, and a conformation where the peptide bond is pointing toward the flavin, approximating the conformation in the semiquinone state. We use single‐crystal spectroscopy to demonstrate that the mixture of conformers is not caused by radiation damage to the crystal. This is the first time that such a mixture of conformers is reported in a wild‐type Fld. We therefore carried out a survey of published Fld structures, which show that several proteins have a pronounced conformational flexibility of this peptide bond. The degree of flexibility seems to be modulated by the presence, or absence, of stabilizing interactions between the peptide bond carbonyl and its surrounding amino acids. We hypothesize that the degree of conformational flexibility will affect the Fld ox/sq redox potential.     

Role of neighboring FMN side chains in the modulation of flavin reduction potentials and in the energetics of the FMN:apoprotein interaction in Anabaena flavodoxin

Flavodoxins (Flds) are electron transfer proteins that carry a noncovalently bound flavin mononucleotide molecule (FMN) as a redox active center. A distinguishing feature of these flavoproteins is the dramatic change in the E(sq/rd) reduction potential of the FMN upon binding to the apoprotein (at pH 8.0, from -269 mV when free in solution to -438 mV in Anabaena Fld). In this study, the contribution of three neighboring FMN residues, Thr56, Asn58, and Asn97, and of three negatively charged surface residues, Glu20, Asp65, and Asp96, to modulate the redox properties of FMN upon its binding to the apoprotein has been investigated. Additionally, the role of these residues in the apoflavodoxin:FMN interaction has been analyzed. Concerning the redox potentials, the most noticeable result was obtained for the Thr56Gly mutant. In this Fld variant, the increased accessibility of FMN leads to an increase of +63 mV in the E(sq/rd) value. On the other hand, a correlation between the electrostatic environment of FMN and the E(sq/rd) has been observed. The more positive residues or the less negative residues present in the surroundings of the FMN N(1) atom, then the less negative the value for E(sq/rd). With regard to FMN binding to apoflavodoxin, breaking of hydrophobic interactions between FMN and residues 56, 58, and 97 seems to increase the K(d) values, especially in the Thr56Gly Fld. Such results suggest that the H-bond network in the FMN environment influences the FMN affinity.     

Nitric-oxide synthase output state. Design and properties of nitric-oxide synthase oxygenase/FMN domain constructs

Mammalian nitric-oxide synthases are large modular enzymes that evolved from independently expressed ancestors. Calmodulin-controlled isoforms are signal generators; calmodulin activates electron transfer from NADPH through three reductase domains to an oxygenase domain. Structures of the reductase unit and its homologs show FMN and FAD in contact but too isolated from the protein surface to permit exit of reducing equivalents. To study states in which FMN/heme electron transfer is feasible, we designed and produced constructs including only oxygenase and FMN binding domains, eliminating strong internal reductase complex interactions. Constructs for all mammalian isoforms were expressed and purified as dimers. All synthesize NO with peroxide as the electron donor at rates comparable with corresponding oxygenase constructs. All bind cofactors nearly stoichiometrically and have native catalytic sites by spectroscopic criteria. Modest differences in electrochemistry versus independently expressed heme and FMN binding domains suggest interdomain interactions. These interactions can be convincingly demonstrated via calmodulin-induced shifts in high spin ferriheme EPR spectra and through mutual broadening of heme and FMNH. radical signals in inducible nitric-oxide synthase constructs. Blue neutral FMN semiquinone can be readily observed; potentials of one electron couple (in inducible nitric-oxide synthase oxygenase FMN, FMN oxidized/semiquinone couple = +70 mV, FMN semiquinone/hydroquinone couple = -180 mV, and heme = -180 mV) indicate that FMN is capable of serving as a one electron heme reductant. The construct will serve as the basis for future studies of the output state for NADPH derived reducing equivalents.     

Structural insight into the high reduction potentials observed for Fusobacterium nucleatum flavodoxin

Flavodoxins are small flavin mononucleotide (FMN)‐containing proteins that mediate a variety of electron transfer processes. The primary sequence of flavodoxin from Fusobacterium nucleatum, a pathogenic oral bacterium, is marked with a number of distinct features including a glycine to lysine (K13) substitution in the highly conserved phosphate‐binding loop (T/S‐X‐T‐G‐X‐T), variation in the aromatic residues that sandwich the FMN cofactor, and a more even distribution of acidic and basic residues. The E_ox/sq (oxidized/semiquinone; ?43 mV) and E_sq/hq (semiquinone/hydroquinone; ?256 mV) are the highest recorded reduction potentials of known long‐chain flavodoxins. These more electropositive values are a consequence of the apoprotein binding to the FMN hydroquinone anion with ~70‐fold greater affinity compared to the oxidized form of the cofactor. Inspection of the FnFld crystal structure revealed the absence of a hydrogen bond between the protein and the oxidized FMN N5 atom, which likely accounts for the more electropositive E_ox/sq. The more electropositive E_sq/hq is likely attributed to only one negatively charged group positioned within 12 Å of the FMN N1. We show that natural substitutions of highly conserved residues partially account for these more electropositive reduction potentials.     

Regulation of the catalytic function of coagulation factor VIIa by a conformational linkage of surface residue Glu 154 to the active site

Coagulation factor VIIa is an allosterically regulated trypsin-like serine protease that initiates the coagulation pathways upon complex formation with its cellular receptor and cofactor tissue factor (TF). The analysis of a conformation-sensitive monoclonal antibody directed to the macromolecular substrate exosite in the VIIa protease domain demonstrated a conformational link from this exosite to the catalytic cleft that is independent of cofactor-induced allosteric changes. In this study, we identify Glu 154 as a critical surface-exposed exosite residue side chain that undergoes conformational changes upon active site inhibitor binding. The Glu 154 side chain is important for hydrolysis of scissile bond mimicking peptidyl p-nitroanilide substrates, and for inhibition of VIIa's amidolytic function upon antibody binding. This exosite residue is not linked to the catalytic cleft residue Lys 192 which plays an important role in thrombin's allosteric coupling to exosite I. Allosteric linkages between VIIa's active site and the cofactor binding site or between the cofactor binding site and the macromolecular substrate exosite were not influenced by mutation of Glu 154. Glu 154 thus only influences the linkage of the macromolecular substrate binding exosite to the catalytic center. These data provide novel evidence that allosteric regulation of VIIa's catalytic function involves discrete and independent conformational linkages and that allosteric transitions in the VIIa protease domain are not globally coupled.     

Another look at the molecular mechanism of the resistance of H5N1 influenza A virus neuraminidase (NA) to oseltamivir (OTV)

In the context of a recent pandemic threat by the worldwide spread of H5N1 avian influenza, the high resistance of H5N1 virus to the most widely used commercial drug, oseltamivir (Tamiflu), is currently an important research topic. Herein, molecular bases of the mechanism of H5N1 NA resistance to oseltamivir were elucidated using a computational approach in a systematic fashion. Using the crystal structure of the complex of H5N1 NA with OTV (PDB ID: 2hu0) as the starting point, the question, how mutations at His274 by both smaller side chain (Gly, Ser, Asn, Gln) and larger side chain (Phe, Tyr) residues influence the sensitivity of N1 to oseltamivir, was addressed and correlated with the experimental data. The smaller side chain residue mutations of His274 resulted in slightly enhanced or unchanged NA sensitivity to OTV, while His274Phe and His274Tyr reduced the susceptibility of OTV to N1. In contrast to the binding free energies, the net charges of Glu276 and Arg224, making charge-charge interactions with Glu276, were established to be more sensitive to detecting subtle conformational differences induced at the key residue Glu276 by the His274X mutations. This study provides deeper insights into the possibility of developing viable drug-resistant mutants.     

Cloning, sequencing and expression of the gene for flavodoxin from Megasphaera elsdenii and the effects of removing the protein negative charge that is closest to N(1) of the bound FMN.

The gene for the electron-transfer protein flavodoxin has been cloned from Megasphaera elsdenii using the polymerase chain reaction. The recombinant gene was sequenced, expressed in an Escherichia coli expression system, and the recombinant protein purified and characterized. With the exception of an additional methionine residue at the N-terminus, the physico-chemical properties of the protein, including its optical spectrum and oxidation-reduction properties, are very similar to those of native flavodoxin. A site-directed mutant, E60Q, was made to investigate the effects of removing the negatively charged group that is nearest to N(1) of the bound FMN. The absorbance maximum in the visible region of the bound flavin moves from 446 to 453 nm. The midpoint oxidation-reduction potential at pH 7 for reduction of oxidized flavodoxin to the semiquinone E2 becomes more negative, decreasing from -114 to -242 mV; E1, the potential for reduction of semiquinone to the hydroquinone, becomes less negative, increasing from -373 mV to -271 mV. A redox-linked pKa associated with the hydroquinone is decreased from 5.8 to < or = 4.3. The spectra of the hydroquinones of wild-type and mutant proteins depend on pH (apparent pKa values of 5.8 and < or = 5.2, respectively). The complexes of apoprotein and all three redox forms of FMN are much weaker for the mutant, with the greatest effect occurring when the flavin is in the semiquinone form. These results suggest that glutamate 60 plays a major role in control of the redox properties of M. elsdenii flavodoxin, and they provide experimental support to an earlier proposal that the carboxylate on its side-chain is associated with the redox-linked pKa of 5.8 in the hydroquinone.     

A conserved histidine in vertebrate-type ferredoxins is critical for redox-dependent dynamics

Adrenodoxin (Adx) belongs to the family of Cys(4)Fe(2)S(2) vertebrate-type ferredoxins that shuttle electrons from NAD(P)H-dependent reductases to cytochrome P450 enzymes. The vertebrate-type ferredoxins contain a conserved basic residue, usually a histidine, adjacent to the third cysteine ligand of the Cys(4)Fe(2)S(2) cluster. In bovine Adx the side chain of this residue, His 56, is involved in a hydrogen-bonding network within the domain of Adx that interacts with redox partners. It has been proposed that this network acts as a mechanical link between the metal cluster binding site and the interaction domain, transmitting redox-dependent conformational or dynamical changes from the cluster binding loop to the interaction domain. H/D exchange studies indicate that oxidized Adx (Adx(o)) is more dynamic than reduced Adx (Adx(r)) on the kilosecond time scale in many regions of the protein, including the interaction domain. Dynamical differences on picosecond to nanosecond time scales between the oxidized (Adx(o)) and reduced (Adx(r)) adrenodoxin were probed by measurement of (15)N relaxation parameters. Significant differences between (15)N R(2) rates were observed for all residues that could be measured, with those rates being faster in Adx(o) than in Adx(r). Two mutations of His 56, H56R and H56Q, were also characterized. No systematic redox-dependent differences between (15)N R(2) rates or H/D exchange rates were observed in either mutant, indicating that His 56 is required for the redox-dependent behavior observed in WT Adx. Comparison of chemical shift differences between oxidized and reduced H56Q and H56R Adx confirms that redox-dependent changes are smaller in these mutants than in the wild-type Adx.     

Functional role of the conserved active site proline of triosephosphate isomerase

The importance of the fully conserved active site proline, Pro168, for the reaction mechanism of triosephosphate isomerase (TIM) has been investigated by studying the enzymatic and crystallographic properties of the P168A variant of trypanosomal TIM. In TIM, Pro168 follows the key catalytic residue Glu167, situated at the beginning of the flexible active site loop (loop 6). Turnover numbers of the P168A variant for its substrates are reduced approximately 50-fold, whereas the Km values are approximately 2 times lower. The affinity of the P168A variant for the transition state analogue 2-phosphoglycolate (2PG) is reduced 5-fold. The crystal structures of unliganded and liganded (2PG) P168A show that the phosphate moiety of 2PG is bound similarly as in wild-type TIM, whereas the interactions of the carboxylic acid moiety with the side chain of the catalytic Glu167 differ. The unique properties of the proline side chain at position 168 are required to transmit ligand binding to the conformational change of Glu167: the side chain of Glu167 flips from the inactive swung-out to the active swung-in conformation on ligand binding in wild-type TIM, whereas in the mutant this conformational change does not occur. Further structural comparisons show that in the wild-type enzyme the concerted movement of loop 6 and loop 7 from unliganded-open to liganded-closed appears to be facilitated by the interactions of the phosphate moiety with loop 7. Apparently, the rotation of 90 degrees of the Gly211-Gly212 peptide plane of loop 7 plays a key role in this concerted movement.     

Engineering conformational flexibility in the lactose permease of Escherichia coli: use of glycine-scanning mutagenesis to rescue mutant Glu325-->Asp

Lactose/H(+) symport by lactose permease of Escherichia coli involves interactions between four irreplaceable charged residues in transmembrane helices that play essential roles in H(+) translocation and coupling [Glu269 (helix VIII) with His322 (helix X) and Arg302 (helix IX) with Glu325 (helix X)], as well as Glu126 (helix IV) and Arg144 (helix V) which are obligatory for substrate binding. The conservative mutation Glu325-->Asp causes a 10-fold reduction in the V(max) for active lactose transport and markedly decreased lactose-induced H(+) influx with no effect on exchange or counterflow, neither of which involves H(+) symport. Thus, shortening the side chain may weaken the interaction of the carboxyl group at position 325 with the guanidino group of Arg302. Therefore, Gly-scanning mutagenesis of helices IX and X and the intervening loop was employed systematically with mutant Glu325-->Asp in an effort to rescue function by introducing conformational flexibility between the two helices. Five Gly replacement mutants in the Glu325-->Asp background are identified that exhibit significantly higher transport activity. Furthermore, mutant Val316-->Gly/Glu325-->Asp catalyzes active transport, efflux, and lactose-induced H(+) influx with kinetic properties approaching those of wild-type permease. It is proposed that introduction of conformational flexibility at the interface between helices IX and X improves juxtapositioning between Arg302 and Asp325 during turnover, thereby allowing more effective deprotonation of the permease on the inner surface of the membrane [Sahin-Tóth, M., Karlin, A., and Kaback, H. R. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 10729-10732.     

Redox properties of the isolated flavin mononucleotide- and flavin adenine dinucleotide-binding domains of neuronal nitric oxide synthase

Electron transfer through neuronal nitric oxide synthase (nNOS) is regulated by the reversible binding of calmodulin (CaM) to the reductase domain of the enzyme, the conformation of which has been shown to be dependent on the presence of substrate, NADPH. Here we report the preparation of the isolated flavin mononucleotide (FMN)-binding domain of nNOS with bound CaM and the electrochemical analysis of this and the isolated flavin adenine dinucleotide (FAD)-binding domain in the presence and absence of NADP(+) and ADP (an inhibitor). The FMN-binding domain was found to be stable only in the presence of bound CaM/Ca(2+), removal of which resulted in precipitation of the protein. The FMN formed a kinetically stabilized blue semiquinone with an oxidized/semiquinone reduction potential of -179 mV. This is 80 mV more negative than the potential of the FMN in the isolated reductase domain, that is, in the presence of the FAD-binding domain. The FMN semiquinone/hydroquinone redox couple was found to be similar in both constructs. The isolated FAD-binding domain, generated by controlled proteolysis of the reductase domain, was found to have similar FAD reduction potentials to the isolated reductase domain. Both formed a FAD-hydroquinone/NADP(+) charge-transfer complex with a long-wavelength absorption band centered at 780 nm. Formation of this complex resulted in thermodynamic destabilization of the FAD semiquinone relative to the hydroquinone and a 30 mV increase in the FAD semiquinone/hydroquinone reduction potential. Binding of ADP, however, had little effect. The possible role of the nicotinamide/FADH(2) stacking interaction in controlling electron transfer and its likely dependence on protein conformation are discussed.     

The intraflavin hydrogen bond in human electron transfer flavoprotein modulates redox potentials and may participate in electron transfer.

Electron-transfer flavoprotein (ETF) serves as an intermediate electron carrier between primary flavoprotein dehydrogenases and terminal respiratory chains in mitochondria and prokaryotic cells. The three-dimensional structures of human and Paracoccus denitrificans ETFs determined by X-ray crystallography indicate that the 4'-hydroxyl of the ribityl side chain of FAD is hydrogen bonded to N(1) of the flavin ring. We have substituted 4'-deoxy-FAD for the native FAD and investigated the analog-containing ETF to determine the role of this rare intra-cofactor hydrogen bond. The binding constants for 4'-deoxy-FAD and FAD with the apoprotein are very similar, and the energy of binding differs by only 2 kJ/mol. The overall two-electron oxidation-reduction potential of 4'-deoxy-FAD in solution is identical to that of FAD. However, the potential of the oxidized/semiquinone couple of the ETF containing 4'-deoxy-FAD is 0.116 V less than the oxidized/semiquinone couple of the native protein. These data suggest that the 4'-hydoxyl-N(1) hydrogen bond stabilizes the anionic semiquinone in which negative charge is delocalized over the N(1)-C(2)O region. Transfer of the second electron to 4'-deoxy-FAD reconstituted ETF is extremely slow, and it was very difficult to achieve complete reduction of the flavin semiquinone to the hydroquinone. The turnover of medium chain acyl-CoA dehydrogenase with native ETF and ETF containing the 4'-deoxy analogue was essentially identical when the reduced ETF was recycled by reduction of 2,6-dichlorophenolindophenol. However, the steady-state turnover of the dehydrogenase with 4'-deoxy-FAD was only 23% of the turnover with native ETF when ETF semiquinone formation was assayed directly under anaerobic conditions. This is consistent with the decreased potential of the oxidized semiquinone couple of the analog-containing ETF. ETF containing 4'-deoxy-FAD neither donates to nor accepts electrons from electron-transfer flavoprotein ubiquinone oxidoreductase (ETF-QO) at significant rates (相似文献