RESUMEN
The four-residue reverse turn -Met56-Gly-Asp-Glu59- in the Clostridium beijerinckii flavodoxin provides the majority of the critical interactions with the isoalloxazine ring of the flavin mononucleotide (FMN) cofactor that contribute to the binding and the differential stabilization of its three redox states. Direct side chain contacts include the sulfur-ring interaction of Met56, which primarily influences the oxidized and hydroquinone states, and the hydrogen bond by Glu59 with the N3H, which directly (and indirectly through its "anchoring" function) influences all three states to various extents. Involving a novel redox-dependent conformational change, the hydrogen bond formed between the carbonyl group of Gly57 and the N5H of the reduced cofactor strongly influences the stability of the semiquinone state. In this study, the sequential elimination of all side chain interactions in various combinations through a systematic alanine-scanning mutagenesis approach was conducted to more completely understand the functional inter-relationships as well as any synergistic interactions that might occur within the loop. In general, additive effects for each side chain on the midpoint potentials for both couples were observed except for the hydroquinone state where some degree of nonadditivity was noted in multiple mutants involving Glu59. The study concluded with the generation of the triple mutant -Ala56-Gly-Ala-Ala59- in which all side chain interactions are removed. Gly57 was left unchanged because of its critical conformational contribution. Remarkably, this mutant retained the ability to bind the FMN and to thermodynamically stabilize the semiquinone state despite the absence of all side chain interactions. Collectively, these observations emphasize the overriding importance of the main chain interactions with the N5H of the FMN and the associated redox-dependent conformational change in this loop and leaves little doubt as to its role in the thermodynamic stabilization of the neutral semiquinone state of the FMN cofactor.
Asunto(s)
Clostridium/química , Flavodoxina/química , Alanina/química , Sustitución de Aminoácidos , Mononucleótido de Flavina/metabolismo , Flavodoxina/genética , Flavodoxina/metabolismo , Ácido Glutámico/genética , Metionina/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Estructura Secundaria de ProteínaRESUMEN
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.
Asunto(s)
Clostridium/química , Mononucleótido de Flavina/química , Flavodoxina/química , Sustitución de Aminoácidos/genética , Sitios de Unión/genética , Mononucleótido de Flavina/metabolismo , Flavodoxina/genética , Flavodoxina/metabolismo , Ácido Glutámico/genética , Enlace de Hidrógeno , Hidroquinonas/química , Modelos Químicos , Mutagénesis Sitio-Dirigida , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular/métodos , Oxidación-Reducción , Potenciometría , Protones , Temperatura , TermodinámicaRESUMEN
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.
Asunto(s)
Desulfovibrio vulgaris/química , Flavodoxina/química , Riboflavina/química , Sitios de Unión , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Isótopos de Nitrógeno , Oxidación-Reducción , TemperaturaRESUMEN
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.
Asunto(s)
Flavodoxina/química , Conformación Proteica , Proteínas Bacterianas/química , Clostridium , Oxidación-Reducción , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Clostridium/química , Mononucleótido de Flavina/química , Flavina-Adenina Dinucleótido/análogos & derivados , Flavodoxina/química , Ácido Glutámico/química , Dicroismo Circular , Clostridium/genética , Clostridium/metabolismo , Coenzimas/química , Coenzimas/genética , Coenzimas/metabolismo , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/metabolismo , Flavodoxina/genética , Flavodoxina/metabolismo , Ácido Glutámico/metabolismo , Glutamina/genética , Glutamina/metabolismo , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Hidroquinonas/metabolismo , Mutagénesis Sitio-Dirigida , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Unión Proteica/genética , Protones , Espectrofotometría Ultravioleta , Temperatura , TermodinámicaRESUMEN
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.
Asunto(s)
Apoproteínas/química , Apoproteínas/genética , Benzoquinonas/química , Mononucleótido de Flavina/química , Flavodoxina/química , Flavodoxina/genética , Glicina/genética , Protones , Sustitución de Aminoácidos/genética , Apoproteínas/metabolismo , Benzoquinonas/metabolismo , Sitios de Unión/genética , Clostridium/química , Clostridium/genética , Mononucleótido de Flavina/metabolismo , Flavodoxina/metabolismo , Enlace de Hidrógeno , Hidroquinonas/química , Hidroquinonas/metabolismo , Mutagénesis Sitio-Dirigida , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular/métodos , Oxidación-Reducción , Conformación Proteica , TemperaturaRESUMEN
Flavodoxins are small electron transferases that participate in low-potential electron transfer pathways. The flavodoxin protein is able to separate the two redox couples of the noncovalently bound flavin mononucleotide (FMN) cofactor through the differential thermodynamic stabilization or destabilization of each of its redox states. In the flavodoxin from Clostridium beijerinckii, the sulfur atom of methionine 56 is in direct contact with the re or inner face of the isoalloxazine ring of the FMN cofactor. In this study, evidence was sought for a possible role for sulfur-aromatic (flavin) interactions in the regulation of one-electron reduction potentials in flavoproteins. Met56 was systematically replaced with all the naturally occurring aliphatic amino acids by site-directed mutagenesis. Replacement of Met56 with alanine or glycine increased the midpoint potentials at pH 7 for the oxidized-semiquinone couple by up to 20 mV compared to that of the wild type, while replacement by the longer chain aliphatic residues decreased the midpoint potential by >30 mV. The midpoint potential for the semiquinone-hydroquinone couple was less negative than that for the wild type for all the mutants, increasing by as much as 90 mV for the M56I mutant. For the M56A mutant, the loss of approximately 0.5 kcal/mol in the binding energy for oxidized FMN and an increase of 1. 6 kcal/mol for the flavin hydroquinone, relative to that of the wild type, are responsible for the observed changes in the midpoint potentials. The stability of the semiquinone complex of this mutant was not affected. The one-election reduction potentials for the M56L, M56I, and M56V mutants are also influenced by the differential stabilization of the three redox states; however, the semiquinone complex was significantly less stable in these proteins. These differences are likely the consequence of the introduction of additional steric factors and an apparent structural preference for a smaller or more flexible side chain at this position in the semiquinone complex. While the other factors may contribute, it is argued that the results obtained for the entire group of mutants are consistent with the elimination of important sulfur-flavin interactions that contribute in part to the stabilization of the oxidized and destabilization of the hydroquinone states of the cofactor in this flavodoxin. The results of this study also demonstrate unequivocally the functional importance of this methionine residue and that it is unique among the aliphatic amino acids in its capacity to generate the physiologically relevant low reduction potential exhibited by the C. beijerinckii flavodoxin.
Asunto(s)
Sustitución de Aminoácidos/genética , Clostridium/metabolismo , Flavinas/metabolismo , Flavodoxina/metabolismo , Metionina/metabolismo , Azufre/metabolismo , Escherichia coli/genética , Mononucleótido de Flavina/metabolismo , Flavodoxina/biosíntesis , Flavodoxina/genética , Metionina/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/químicaRESUMEN
A hypothetical model for electron transfer complex between cytochrome c3 and the flavodoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris has been proposed, based on electrostatic potential field calculations and NMR data [Stewart, D. E., LeGall, J. , Moura, I., Moura, J. J. G., Peck, H. D., Jr., Xavier, A. V., Weiner, P. K., & Wampler, J. E. (1988) Biochemistry 27, 2444-2450]. This modeled complex relies primarily on the formation of five ion pairs between lysine residues of the cytochrome and acidic residues surrounding the flavin mononucleotide cofactor of the flavodoxin. In this study, the role of several acidic residues of the flavodoxin in the formation of this complex and in electron transfer between these two proteins was evaluated. A total of 17 flavodoxin mutants were studied in which 10 acidic amino acids--Asp62, Asp63, Glu66, Asp69, Asp70, Asp95, Glu99, Asp106, Asp127, and Asp129--had been permanently neutralized either individually or in various combinations by substitution with their amide amino acid equivalent (i.e., asparate to asparagine, glutamate to glutamine) through site-directed mutagenesis. The kinetic data for the transfer of electrons from reduced cytochrome c3 to the various flavodoxin mutants do not conform well to a simple bimolecular mechanism involving the formation of an intermediate electron transfer complex. Instead, a minimal electron transfer mechanism is proposed in which an initial complex is formed that is stabilized by intermolecular electrostatic interactions but is relatively inefficient in terms of electron transfer. This step is followed by a rate-limiting reorganization of that complex leading to efficient electron transfer. The apparent rate of this reorganization step was enhanced by the disruption of the initial electrostatic interactions through the neutralization of certain acidic amino acid residues leading to faster overall observed electron transfer rates at low ionic strengths. Of the five acidic residues involved in ion pairing in the modeled complex proposed by Stewart et al. (1988), the kinetic data strongly implicate Asp62, Glu66, and Asp95 in the formation of the electrostatic interactions that control electron transfer. Less certainty is provided by this study for the involvement of Asp69 and Asp129, although the data do not exclude their participation. It was not possible to determine whether the modeled complex represents the optimal configuration for electron transfer obtained after the reorganization step or actually represents the initial complex. The data do provide evidence for the importance of electrostatic interactions in electron transfer between these two proteins and for the existence of alternative binding modes involving acidic residues on the surface of the flavodoxin other than those proposed in that model.
Asunto(s)
Aminoácidos Dicarboxílicos/química , Grupo Citocromo c/química , Desulfovibrio vulgaris/química , Flavodoxina/química , Aminoácidos Dicarboxílicos/genética , Asparagina/genética , Ácido Aspártico/genética , Desulfovibrio vulgaris/enzimología , Desulfovibrio vulgaris/genética , Electroquímica , Transporte de Electrón/genética , Flavodoxina/biosíntesis , Flavodoxina/genética , Regulación Bacteriana de la Expresión Génica , Ácido Glutámico/genética , Glutamina/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Concentración OsmolarRESUMEN
Flavodoxin from Desulfovibrio vulgaris is a low molecular weight (15 000 Da) acidic flavoprotein that contains a single flavin mononucleotide (FMN) cofactor. A distinguishing feature of the flavodoxin family is the exceptionally low midpoint potential of the semiquinone/hydroquinone couple. Tyrosine-98, which flanks the outer or si face of the FMN, plays an important role in establishing the oxidation-reduction properties of the bound cofactor as demonstrated by the substitution of a number of amino acids at this position [Swenson, R. P., & Krey, G. D. (1994) Biochemistry 33, 8505-8514]. The midpoint potential for the semiquinone/hydroquinone couple increases substantially when basic residues are introduced at this position. The pH dependency in the Y98H mutant is consistent with a redox-linked ionization model in which the favorable electrostatic coupling between the imidazolium cation and the flavin hydroquinone anion is responsible for the higher potential. Such a model predicts an increase in the pKa of 1.5 units for His98 upon complete reduction of the FMN. In this study, proton nuclear magnetic resonance spectroscopy was used to directly determine the intrinsic pKa of His98 as a function of the redox state of the cofactor in this flavodoxin. Values for the pKa of His98 in the oxidized and fully reduced flavodoxin are 7.02 +/- 0.08 and 8.43 +/- 0.11, respectively, an increase in the pKa by 1.41 units, which conforms with the previous prediction. These results provide direct experimental proof of the redox-linked ionization of this residue and provides further evidence of the crucial role of electrostatic interactions, in this case, in the stabilization of the flavin hydroquinone anion. This phenomenon may represent a general mechanism in the modulation of the reduction potential of the flavin cofactor within flavoenzymes in which ionizable groups such as histidine in the active center change ionization states during the catalytic cycle.
Asunto(s)
Flavodoxina/química , Histidina/química , Sitios de Unión , Catálisis , Desulfovibrio vulgaris , Escherichia coli , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Oxidación-ReducciónRESUMEN
X-ray analyses of wild-type and mutant flavodoxins from Clostridium beijerinckii show that the conformation of the peptide Gly57-Asp58, in a bend near the isoalloxazine ring of FMN, is correlated with the oxidation state of the FMN prosthetic group. The Gly-Asp peptide may adopt any of three conformations: trans O-up, in which the carbonyl oxygen of Gly57 (O57) points toward the flavin ring; trans O-down, in which O57 points away from the flavin; and cis O-down. Interconversions among these conformers that are linked to oxidation-reduction of the flavin can modulate the redox potentials of bound FMN. In the semiquinone and reduced forms of the protein, the Gly57-Asp58 peptide adopts the trans O-up conformation and accepts a hydrogen bond from the flavin N5H [Smith, W. W., Burnett, R. M., Darling, G. D., & Ludwig, M. L. (1977) J. Mol. Biol. 117, 195-225; Ludwig, M. L., & Luschinsky, C. L. (1992) in Chemistry and Biochemistry of Flavoenzymes III (Müller, F., Ed.) pp 427-466, CRC Press, Boca Raton, FL]. Analyses reported in this paper confirm that, in crystals of wild-type oxidized C. beijerinckii flavodoxin, the Gly57-Asp58 peptide adopts the O-down orientation and isomerizes to the cis conformation. This cis form is preferentially stabilized in the crystals by intermolecular hydrogen bonding to Asn137. Structures for the mutant Asn137Ala indicate that a mixture of all three conformers, mostly O-down, exists in oxidized C. beijerinckii flavodoxin in the absence of intermolecular hydrogen bonds. Redox potentials have been manipulated by substitutions that alter the conformational energies of the bend at 56M-G-D-E. The mutation Asp58Pro was constructed to study a case where energies for cis-trans conversion would be different from that of wild type. Intermolecular interactions with Asn137 are precluded in the crystal, yet Gly57-Pro58 is cis, and O-down, when the flavin is oxidized. Reduction of the flavin induces rearrangement to the trans O-up conformation. Redox potential shifts reflect the altered energies associated with the peptide rearrangement; E(ox/sq) decreases by approximately 60 mV (1.3 kcal/mol). Further, the results of mutation of Gly57 agree with predictions that a side chain at residue 57 should make addition of the first electron more difficult, by raising the energy of the O-up conformer that forms when the flavin is reduced to its semiquinone state. The ox/sq potentials in the mutants Gly57Ala, Gly57Asn, and Gly57Asp are all decreased by approximately 60 mV (1.3 kcal/mol). Introduction of the beta-branched threonine side chain at position 57 has much larger effects on the conformations and potentials. The Thr57-Asp58 peptide adopts a trans O-down conformation when the flavin is oxidized; upon reduction to the semiquinone, the 57-58 peptide rotates to a trans O-up conformation resembling that found in the wild-type protein. Changes in FMN-protein interactions and in conformational equilibria in G57T combine to decrease the redox potential for the ox/sq equilibrium by 180 mV (+4.0 kcal/mol) and to increase the sq/hq potential by 80 mV (-1.7 kcal/mol). A thermodynamic scheme is introduced as a framework for rationalizing the properties of wild-type flavodoxin and the effects of the mutations.
Asunto(s)
Flavodoxina/química , Secuencia de Aminoácidos , Clostridium , Cristalografía por Rayos X , Mononucleótido de Flavina/metabolismo , Flavinas/metabolismo , Flavodoxina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Conformación Proteica , Espectrofotometría Atómica , Espectrofotometría Ultravioleta , Relación Estructura-ActividadRESUMEN
Flavodoxins are typified by the very low one-electron reduction potential for the semiquinone/hydroquinone couple (Esq/hq) of the flavin mononucleotide (FMN) cofactor. In the Desulfovibrio vulgaris flavodoxin, the elimination of the side chain of Tyr98, which flanks the outer or si face of the flavin, through the Y98A mutation results in a substantial increase in Esq/hq of 139 mV, representing about one-half of the total shift in Esq/hq in this flavodoxin [Swenson, R. P., & Krey, G. D. (1994) Biochemistry 33, 8505-8514]. The extent to which this large effect was the result of the elimination of unfavorable coplanar aromatic stacking interactions or to the greater solvent exposure of the flavin ring was not known. The significance of the latter effect was heightened by the characterization of the Fld+6 mutant which demonstrated that the unfavorable interaction between the negative electrostatic environment provided by the asymmetric clustering of acidic residues surrounding the cofactor and the FMN hydroquinone anion is responsible for about one-third of the total decrease in Esq/hq in this flavodoxin [Zhou, Z., & Swenson, R. P. (1995) Biochemistry 34, 3183-3192]. In this study, a flavodoxin mutant was generated in which an alanine was substituted for Tyr98 while at the same time the negative electrostatic surface was partially neutralized by the substitution of the six acidic amino acid residues with their amide equivalents. The Esq/hq value of this mutant was found to have increased by 221 mV relative to wild type, which accounts for 70-80% of the total shift in Esq/hq in this flavodoxin. This increase is very similar to the sum of the individual changes in Esq/hq introduced independently in the Y98A and Fld+6 mutants. The similarity in the magnitude of the effect of the neutralization of the six acidic residues in the context of an alanine residue at position 98 (Y98A) relative to an aromatic tyrosine residue (wild type) suggests that the increase in Esq/hq observed for the Y98A mutant is more likely due to the elimination of unfavorable pi-pi interactions between Tyr98 and the FMN hydroquinone rather than to the increased solvent exposure of the flavin. This study provides further support for the concept that the cumulative effect of the unfavorable electrostatic interactions introduced by coplanar aromatic or pi-pi stacking interactions and the negative electrostatic environment of the FMN binding site is a major determinant of the low one-electron reduction potential of the flavodoxin.
Asunto(s)
Desulfovibrio vulgaris/metabolismo , Mononucleótido de Flavina/metabolismo , Flavodoxina/química , Flavodoxina/metabolismo , Estructura Terciaria de Proteína , Sitios de Unión , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Mutación Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Electricidad Estática , TirosinaRESUMEN
Two mutants of the Desulfovibrio vulgaris flavodoxin, T12H and N14H, were generated which, for the first time, place a basic residue within the normally neutral 5'-phosphate binding loop of the flavin mononucleotide cofactor binding site found in all flavodoxins. These histidine residues were designed to form an ion pair with the dianionic 5'-phosphate, either altering its ionization state or offsetting its negative charge to allow evaluation of the magnitude of its electrostatic effect on the redox properties of the cofactor. The midpoint potential for the oxidized/semiquinone couple was not significantly altered in either mutant. However, the midpoint potentials for the semiquinone/hydroquinone couple (Esq/hq) were less negative than that of the wild type, increasing by 28 and 15 mV relative to that of the wild type for the T12H and N14H mutants, respectively, at pH 6. 31P NMR spectroscopy suggests that, just as for wild type, the phosphate group in each mutant does not change its ionization state between pH 6 and 8. Therefore, the small increases in midpoint potential must be linked to the protonation of the histidine residues, either through favorable interactions with the anionic hydroquinone or by the partial compensation of the charge on the 5'-phosphate. Values for the pKa of His12 and His14 in the oxidized flavodoxin were determined by 1H NMR spectroscopy to be 6.71 and 6.93, respectively, which are only modestly elevated relative to the average value for histidines in proteins. This suggests that the histidines do not form strong ion-pairing interactions with the phosphate and/or that the effective charge on the 5'-phosphate may be substantially less than the reported formal dianionic charge. Either way, the data provide evidence for the rather weak electrostatic interaction between a charged group at this site and the anionic flavin hydroquinone. In contrast, Esq/hq reported for the apoflavodoxin-riboflavin complex, which lacks the 5'-phosphate group, is 180 mV less negative than that of the native flavodoxin. The re-evaluation of the redox and cofactor binding properties of the riboflavin complex generated values for the dissociation constants for the riboflavin complex in the oxidized, semiquinone, and hydroquinone oxidation states that are 2100-, 63000-, and 54-fold higher, respectively, than that for the naturally occurring flavin mononucleotide complex. The large redox potential shifts observed for both redox couples in the riboflavin complex are primarily the consequence of a decreased stabilization of the semiquinone rather than the result of the absence of the negative charge of the 5'-phosphate. It is concluded from this study that the negative charge on the phosphate group of the cofactor does not play a disproportionate role in decreasing Esq/hq, at most contributing equivalently with the acidic amino acid residues clustered around the flavin to an unfavorable electrostatic environment for the formation of the flavin hydroquinone anion.
Asunto(s)
Desulfovibrio vulgaris/química , Mononucleótido de Flavina/química , Flavodoxina/metabolismo , Fosfatos/química , Apoproteínas/química , Sitios de Unión , Mononucleótido de Flavina/metabolismo , Flavodoxina/química , Flavodoxina/genética , Histidina/química , Histidina/genética , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Riboflavina/química , EspectrofotometríaRESUMEN
Sequence-specific 1H and 15N resonance assignments have been made for 137 of the 146 nonprolyl residues in oxidized Desulfovibrio desulfuricans [Essex 6] flavodoxin. Assignments were obtained by a concerted analysis of the heteronuclear three-dimensional 1H-15N NOESY-HMQC and TOCSY-HMQC data sets, recorded on uniformly 15N-enriched protein at 300 K. Numerous side-chain resonances have been partially or fully assigned. Residues with overlapping 1HN chemical shifts were resolved by a three-dimensional 1H-15N HMQC-NOESY-HMQC spectrum. Medium- and long-range NOEs, 3JNH alpha coupling constants, and 1HN exchange data indicate a secondary structure consisting of five parallel beta-strands and four alpha-helices with a topology similar to that of Desulfovibrio vulgaris [Hildenborough] flavodoxin. Prolines at positions 106 and 134, which are not conserved in D. vulgaris flavodoxin, contort the two C-terminal alpha-helices.
Asunto(s)
Flavodoxina/química , Secuencia de Aminoácidos , Desulfovibrio/química , Desulfovibrio/genética , Desulfovibrio vulgaris/química , Desulfovibrio vulgaris/genética , Flavodoxina/genética , Hidrógeno/química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Oxidación-Reducción , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Soluciones , Especificidad de la EspecieRESUMEN
The flavodoxin from Desulfovibrio vulgaris (Hildenborough) is a member of a family of small, acidic proteins that contain a single noncovalently bound flavin mononucleotide (FMN) cofactor. These proteins function as low-potential one-electron transferases in bacteria. A distinguishing feature of these flavoproteins is the dramatic decrease in the midpoint potential of the semiquinone/hydroquinone couple of the FMN upon binding to the apoprotein (-172 mV for FMN free in solution versus -443 mV when bound), a perturbation thought to be essential for physiological function. The structural basis of this phenomenon is not yet thoroughly understood. In this study, the contribution of six acidic residues (Asp62, Asp63, Glu66, Asp95, Glu99, and Asp106) to the perturbation of the redox properties of the cofactor has been investigated. These residues are clustered about the FMN binding site within 13 A of the N(1) atom of the cofactor. Using oligonucleotide-directed mutagenesis, these residues were neutralized in various combinations through the substitution of asparagine for aspartate and glutamine for glutamate. Seventeen mutant flavodoxins were generated in which one to all six acidic residues were systematically neutralized, often in various spatial configurations. There was no obvious correlation between the midpoint potentials for the oxidized/semiquinone couple and general electrostatic environment, although some differences were noted. However, the midpoint potential for the semiquinone/hydroquinone couple for each of the mutants was less negative than that of the wild type. These increases are strongly correlated with the number of acid to amide substitutions, with an average contribution of about 15 mV per substitution. Collectively, the unfavorable electrostatic environment provided by these acidic residues accounts for approximately one-third of the large midpoint potential shift for the semiquinone/hydroquinone couple that typifies the flavodoxin family, apparently through the destabilization of the flavin hydroquinone anion.
Asunto(s)
Desulfovibrio vulgaris/química , Flavodoxina/química , Aminoácidos Dicarboxílicos/química , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Desulfovibrio vulgaris/genética , Electroquímica , Escherichia coli/genética , Flavodoxina/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Conformación ProteicaRESUMEN
The genes encoding the two different subunits of the electron transfer flavoprotein (ETF) from the methylotrophic bacterium W3A1 have been identified, cloned, and sequenced. A 0.8-kilobase pair DNA fragment was generated for use as a molecular probe by the amplification of genomic sequences using the polymerase chain reaction and a primer pair with degenerate sequences derived from the NH2-terminal amino acid sequences determined for the ETF subunits purified from W3A1. The screening of a partial genomic minilibrary containing size-selected BamHI-SalI fragments using this probe identified a 2.2-kilobase pair insert containing the complete coding sequences for both W3A1 ETF subunits. The genes are arranged in tandem in the genomic DNA with only 2 bases between the TAG translation termination codon of the small subunit and the ATG translation initiation codon of the large subunit. The deduced amino acid sequences of each of the W3A1 ETF subunits exhibit only approximately 30% identity with the corresponding subunits of the ETF from human, rat, and Paracoccus denitrificans, which as a group are greater than 50% identical. Thus, the ETF from W3A1 may exhibit some unique structural features that, like other differences in some of its physical and functional properties, may distinguish this ETF from others in this family. A highly homologous region near the COOH terminus of the large subunit in all the ETF proteins was found to contain a sequence that matches in several ways the ADP-binding motif of flavoproteins and other dinucleotide-binding proteins, suggesting that the large subunit forms a portion of the FAD (or AMP) binding site in these proteins. Under control of the tac promoter, the cloned ETF subunit genes were co-expressed in Escherichia coli producing the heterodimeric holoprotein with physical, spectral, and electron-accepting properties essentially identical to the ETF isolated from W3A1. The recombinant ETF serves as the electron acceptor for W3A1 trimethylamine dehydrogenase in vitro, accumulating as the air-stable anionic semiquinone in the presence of excess trimethylamine. Fully reduced ETF could not be obtained even after prolonged enzymatic reduction.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Portadoras , Flavoproteínas/genética , Genes Bacterianos , Bacterias Gramnegativas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Humanos , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes/genética , Homología de Secuencia de AminoácidoRESUMEN
Flavodoxins mediate electron transfer at low redox potential between the prosthetic groups of other proteins. Interactions between the protein and the flavin mononucleotide cofactor shift both the oxidized/semiquinone and semiquinone/hydroquinone redox potentials significantly from their free-in-solution values. In order to investigate the possible role that the tyrosine at position 98 plays in this process, we have used heteronuclear three-dimensional NMR spectroscopy to determine the solution conformation of wild-type and four position-98 mutants, Y98W, Y98H, Y98A, and Y98R, of Desulfovibrio vulgaris flavodoxin. Assigned 1H and 15N resonances indicate that the secondary structure and topology of the proteins are identical. However, residues that undergo substantial mutation-induced changes in chemical shift are spread throughout the flavin cofactor binding site. Distance and dihedral angle constraints were used to generate solution structures for the wild-type and mutant proteins. Collectively, the mutant proteins have no gross conformational changes in the flavin binding site. The changes that do occur are minor and result from the different packing interactions required to accommodate the new side chain at position-98. The solvent accessibility and electrostatic nature of the flavin binding site in the mutant proteins are compared to those of the wild-type structure. The structural data support the hypothesis that the very low midpoint of the semiquinone/hydroquinone couple in the wild-type protein is modulated to a large extent by the energetically unfavorable formation of the flavin hydroquinone anion in the apolar environment of the flavin binding site.
Asunto(s)
Desulfovibrio vulgaris/química , Flavodoxina/química , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Estructura Terciaria de Proteína , Proteínas Recombinantes , Relación Estructura-Actividad , Tirosina/químicaRESUMEN
The contributions made by tyrosine-98 in establishing the redox properties of the flavodoxin from Desulfovibrio vulgaris were investigated by substituting a number of amino acids at this position using site-directed mutagenesis. Tyr98, which makes extensive van der Waals contacts with the isoalloxazine ring of the flavin mononucleotide cofactor, is often found in the cofactor binding site of flavodoxins and related flavoproteins. Solution studies suggest that tyrosine may assist in the stabilization of the neutral flavin semiquinone through preferential complex formation relative to the other oxidation states. In this study, the midpoint potentials of the oxidized/semiquinone couple of the Y98W and Y98F mutants were found to be very similar to the wild-type flavodoxin. However, significantly more negative midpoint potentials (by 25-60 mV) were observed in the Y98A, Y98H, and Y98R mutants. These results imply that it is the general apolar environment provided by the aromatic amino acids rather than preferential affinities suggested by solution studies that is at least partially responsible for the thermodynamic stabilization of the neutral flavin semiquinone in this flavodoxin. The midpoint potential of the semiquinone/hydroquinone couple is profoundly dependent on the properties of the amino acid at this position. Compared to phenylalanine, the more electron-rich aromatic side chains of tryptophan and tyrosine decrease the midpoint potential of this couple by 30-40 mV. Greater solvent exposure of the isoalloxazine ring in the Y98A mutant increases the midpoint potential by 140 mV relative to wild type. The positively charged amino acids increase the midpoint potential of this couple by > 180 mV, most probably through favorable electrostatic interactions with the flavin hydroquinone anion. These observations strongly support the proposition that the functional role of the electron-rich, apolar aromatic amino acid residues adjacent to the flavin isoalloxazine ring is to substantially destabilize the flavin hydroquinone anion, resulting in the very low oxidation-reduction potentials for the semiquinone/hydroquinone couple that typify the flavodoxin family.
Asunto(s)
Desulfovibrio vulgaris/química , Mononucleótido de Flavina/metabolismo , Flavodoxina/química , Mutagénesis Sitio-Dirigida , Tirosina/química , Sitios de Unión , Fenómenos Químicos , Química Física , Estabilidad de Medicamentos , Electroquímica , Mononucleótido de Flavina/química , Flavodoxina/genética , Estructura Molecular , Oxidación-Reducción , Quinonas/química , Quinonas/metabolismo , Solventes , Espectrometría de Fluorescencia , Espectrofotometría , Relación Estructura-Actividad , Termodinámica , Tirosina/genéticaRESUMEN
Sequence-specific 1H and 15N resonance assignments have been made for all 145 non-prolyl residues and for the flavin cofactor in oxidized Desulfovibrio vulgaris flavodoxin. Assignments were obtained by recording and analyzing 1H-15N heteronuclear three-dimensional NMR experiments on uniformly 15N-enriched protein, pH 6.5, at 300 K. Many of the side-chain resonances have also been assigned. Observed medium-and long-range NOEs, in combination with 3JNH alpha coupling constants and 1HN exchange data, indicate that the secondary structure consists of a five-stranded parallel beta-sheet and four alpha-helices, with a topology identical to that determined previously by X-ray crystallographic methods. One helix, which is distorted in the X-ray structure, is non-regular in solution as well. Several protein-flavin NOEs, which serve to dock the flavin ligand to its binding site, have also been identified. Based on fast-exchange into 2H2O, the 1HN3 proton of the isoalloxazine ring is solvent accessible and not strongly hydrogen-bonded in the flavin binding site, in contrast to what has been observed in several other flavodoxins. The resonance assignments presented here can form the basis for assigning single-site mutant flavodoxins and for correlating structural differences between wild-type and mutant flavodoxins with altered redox potentials.
Asunto(s)
Proteínas Bacterianas/química , Desulfovibrio vulgaris/química , Flavodoxina/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Hidrógeno , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Oxidación-Reducción , Proteínas Recombinantes de Fusión/química , SolucionesRESUMEN
The structural genes coding for the flavodoxin proteins from two different strains of the sulfate-reducing bacteria Desulfovibrio gigas (ATCC 19364/NCIB 9332 and ATCC 29494/DSM 496) have been identified, cloned and the nucleotide sequence established. The protein sequences derived from the gene from each strain share a sequence identity of 66% with regions directly involved in binding the flavin mononucleotide cofactor being the most homologous. Both aromatic residues that flank the flavin isoalloxazine ring in the crystal structure of the flavodoxin from D. vulgaris, i.e., Trp-60 and Tyr-98, are also present in these flavodoxin proteins. These observations stand in contrast to reports that the flavodoxin from Desulfovibrio gigas contains a single tryptophan residue which is located distant from the flavin binding site. Therefore, the FMN binding site of this flavodoxin is not distinct from the other Desulfovibrio flavodoxins in this regard.