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S G Mayhew

Publications and source records attributed to S G Mayhew.

At least 55 records · Page 3Linked to original sources
PubMed1993

13C-n.m.r. of the cyanylated apoflavodoxin and flavodoxin from Clostridium pasteurianum.

The thiol group of the flavodoxin from Clostridium pasteurianum has been cyanylated in a single step using [cyanato-13C]2-nitro-5-thiocyanatobenzoic acid. This chemical modification increases the dissociation constant of the apoflavodoxin-FMN complex 10-fold from 0.33 +/- 0.15 nM to 2.9 +/- 1.3 nM. The thiocyanate carbons of the cyanylated cysteine residue of apoflavodoxin and flavodoxin had chemical shift values of 114.7 and 112.3 p.p.m. respectively. From these chemical shifts we conclude that the binding of FMN by the cyanylated apoflavodoxin causes a decrease in the polarity and/or hydrogen bonding capacity of the environment of the thiocyanate group. We compare these results with those obtained from similar studies on the cyanylated apoflavodoxin and flavodoxin from Megasphaera elsdenii and we discuss how FMN binding and cyanylation perturb the structures of apoflavodoxins from Megasphaera elsdenii and Clostridium pasteurianum.

Apoproteins↗
PubMed1993

Effects of thiols and mercurials on the periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough).

The H2-oxidation, H2-production and H-3H-exchange activities of the periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough) were almost completely abolished by Hg(II) and the organic mercurials p-chloromercuribenzoate (pCMB) and p-hydroxymercuriphenylsulphonate. The thiol-modifying reagents N-ethylmaleimide, iodoacetate, dithionitrobenzoate and 2-nitro-5-thiocyanobenzoate had no effect on the activities. Kinetic and spectroscopic measurements suggest that inactivation by pCMB involves at least two reactions; a rapid reaction that is reversed by thiols, and a second, slower and irreversible reaction that occurs at high concentrations of the mercurial. The irreversible reaction was associated with loss of visible absorbance, indicative of a disrupted iron sulphur cluster(s). The effects on the H-3H-exchange activity indicate that the reversible modification affects the H2-activating site. Enzyme that had lost activity due to pCMB treatment, or during long-term storage, was reactivated by thiols. This reactivation was followed by a slower irreversible inactivation, as also occurred with native enzyme; the inactivation was O2 dependent and it was partly prevented by catalase, suggesting that H2O2 may be involved.

Chloromercuribenzoates↗
PubMed1993

Homonuclear and heteronuclear NMR studies of oxidized Desulfovibrio vulgaris flavodoxin. Sequential assignments and identification of secondary structure elements.

Recombinant Desulfovibrio vulgaris flavodoxin (molecular mass 16.3 kDa) was produced in Escherichia coli. The oxidized protein has been investigated with a combination of homonuclear and heteronuclear two-dimensional and heteronuclear three-dimensional NMR spectroscopy. Sequence-specific assignment of all backbone and most of the side chain 1H and 15N resonances has been obtained. The secondary structure has been inferred from the pattern of sequential, medium-, and long-range NOEs, together with information about slowly exchanging amide hydrogens and HN-H alpha spin-spin coupling constants. In solution, flavodoxin consists of a five-stranded parallel beta-sheet and four alpha-helices. Residues 3-9, 32-36, 52-58, 87-96, and 123-128 are involved in the beta-sheet whereas the a-helical regions comprise residues 13-28, 69-76, 104-114, and 134-148. Several proton resonances of the bound flavin mononucleotide cofactor have been assigned. NOE contacts between the prosthetic group and the apoprotein have been detected.

Amino Acid Sequence↗
PubMed1992

13C NMR of cyanylated flavodoxin from Megasphaera elsdenii and of thiocyanate model compounds.

Both of the thiol groups of Megasphaera elsdenii flavodoxin have been cyanylated using 13C-enriched cyanide. This chemical modification increases the dissociation constant of the apoflavodoxin-flavin mononucleotide (FMN) complex from 0.4 nM to 2 microM. The thiocyanate carbons of the cyanylated cysteine residues in apoflavodoxin had 13C chemical shifts of 109.4 ppm and 112.2 ppm, which were replaced by signals at 115.5 ppm and 109.6 ppm when FMN was bound. The signals at 109.4 ppm and 112.2 ppm due to the cyanylated apoflavodoxin were unstable at 28 degrees C, and they were slowly replaced signals at 114.5 ppm and 115.3 ppm which are attributed to an inactive form of the apoprotein, which does not bind FMN. At alkaline pH values or after prolonged incubation at neutral pH, the signals at 114.5 ppm and 115.3 ppm were replaced by signals at approximately 171 ppm. On the basis of results obtained with model compounds, the signals at 171 ppm are assigned to the 2-imino carbon of the 2-iminothiazolidine ring formed by the cyclization of the appropriate thiocyanate group. After determining the chemical shift of the thiocyanate carbon of model compounds in a range of solvents, we conclude that the thiocyanate carbons will have a minimal chemical shift of approximately 109 ppm in apolar solvents which do not contain hydrogen bond donors. In water, a more polar hydrogen-bonding solvent, the chemical shift increases to approximately 115 ppm. We also conclude that the chemical shift of a thiocyanate carbon can be used as a probe of its molecular environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1992

Complex formation between ferredoxin and ferredoxin-NADP+ reductase from Anabaena PCC 7119: cross-linking studies.

Ferredoxin-NADP+ reductase and ferredoxin from the cyanobacterium Anabaena PCC 7119 have been covalently cross-linked by incubation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The covalent adduct, which shows a molecular mass consistent with a 1:1 stoichiometry of the two proteins, maintains nearly 60% of the NADPH-cytochrome c reductase activity of the enzyme saturated with ferredoxin and this value is considerably higher than when equimolar amounts of both proteins are assayed. No ternary complexes with Anabaena flavodoxin or horse heart cytochrome c were formed, suggesting that the binding site on the enzyme is the same for ferredoxin and flavodoxin and that ferredoxin-NADP+ reductase and cytochrome c bind at a common site on ferredoxin. In the noncovalent complex, titrated at pH 7, the oxidation-reduction potential of ferredoxin becomes 15 mV more negative and that of ferredoxin-NADP+ reductase 27 mV more positive compared to the proteins alone. When covalently linked, the midpoint potential of the enzyme has a value similar to that in the noncovalent complex, while the ferredoxin potential is 20 mV more positive compared to ferredoxin alone. The changes in redox potentials have been used to estimate the dissociation constants for the interaction of the different redox forms of the proteins, based on the value of 1.21 microM calculated for the oxidized noncovalent complex.

Anabaena↗
PubMed1992

The hydrogen-tritium exchange activity of Megasphaera elsdenii hydrogenase.

The hydrogenase of Megasphaera elsdenii was purified to a specific activity of 350 units/mg. The hydrogen-tritium exchange assay of Hallahan et al. [Hallahan, D.L., Fernandez, V. M., Hatchikian, E. C. and Hall, D. O. (1986) Biochimie (Paris) 68, 49-54] was adapted to allow its use in the study of the M. elsdenii hydrogenase preparation. Under the assay conditions routinely employed, the enzyme's exchange activity was inhibited by Tris/HCl and MgCl2; it was stimulated by ethylene glycol. Maximal activity in this standard assay occurred at pH 7.1. The effect of the concentration of molecular hydrogen (1H2 plus 3H1H) on the exchange activity was studied. The resulting double-reciprocal plot was linear; its slope and its intercepts on the ordinate and abscissa were pH-dependent. The rate equations for a number of models of the exchange activity were derived. Each model gave rise to a linear double-reciprocal plot at constant pH, but none could explain fully the observed effects of varying pH. The experimental data corresponded most closely to the predictions of models in which protons were treated both as substrates and as regulators of the enzyme's activity.

Buffers↗
PubMed1991

Oxidation-reduction potentials of ferredoxin-NADP+ reductase and flavodoxin from Anabaena PCC 7119 and their electrostatic and covalent complexes.

The oxidation-reduction potentials of ferredoxin-NADP+ reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119 were determined by potentiometry. The potentials at pH 7 for the oxidized flavodoxin/flavodoxin semiquinone couple (E2) and the flavodoxin semiquinone/hydroquinone couple (E1) were -212 mV and -436 mV, respectively. E1 was independent of pH above about pH 7, but changed by approximately -60 mV/pH below about pH 6, suggesting that the fully reduced protein has a redox-linked pKa at about 6.1, similar to those of certain other flavodoxins. E2 varied by -50 mV/pH in the range pH 5-8. The redox potential for the two-electron reduction of ferredoxin-NADP+ reductase was -344 mV at pH 7 (delta Em = -30 mV/pH). In the 1:1 electrostatic complex of the two proteins titrated at pH 7, E2 was shifted by +8 mV and E1 was shifted by -25 mV; the shift in potential for the reductase was +4 mV. The potentials again shifted following treatment of the electrostatic complex with a carbodiimide, to covalently link the two proteins. By comparison with the separate proteins at pH 7, E2 for flavodoxin shifted by -21 mV and E1 shifted by +20 mV; the reductase potential shifted by +2 mV. The potentials of the proteins in the electrostatic and covalent complexes showed similar pH dependencies to those of the individual proteins. Qualitatively similar changes occurred when ferredoxin-NADP+ reductase from Anabaena variabilis was complexed with flavodoxin from Azotobacter vinelandii. The shifts in redox potential for the complexes were used with previously determined values for the dissociation constant (Kd) of the electrostatic complex of the two oxidised proteins, in order to estimate Kd values for the interaction of the different redox forms of the proteins. The calculations showed that the electrostatic complexes, formed when the proteins differ in their redox states, are stronger than those formed when both proteins are fully oxidized or fully reduced.

Anabaena↗
PubMed1991

Redox and flavin-binding properties of recombinant flavodoxin from Desulfovibrio vulgaris (Hildenborough).

Flavodoxin from Desulfovibrio vulgaris (Hildenborough) has been expressed at a high level (3-4% soluble protein) in Escherichia coli by subcloning a minimal insert carrying the gene behind the tac promoter of plasmid pDK6. The recombinant protein was readily isolated and its properties were shown to be identical to those of the wild-type protein obtained directly from D. vulgaris, with the exception that the recombinant protein lacks the N-terminal methionine residue. Detailed measurements of the redox potentials of this flavodoxin are reported for the first time. The redox potential, E2, for the couple oxidized flavodoxin/flavodoxin semiquinone at pH 7.0 is -143 mV (25 degrees C), while the value for the flavodoxin semiquinone/flavodoxin hydroquinone couple (E1) at the same pH is -440 mV. The effects of pH on the observed potentials were examined; E2 varies linearly with pH (slope = -59 mV), while E1 is independent of pH at high pH values, but below pH 7.5 the potential becomes less negative with decreasing pH, indicating a redox-linked protonation of the flavodoxin hydroquinone. D. vulgaris apoflavodoxin binds FMN very tightly, with a value of 0.24 nM for the dissociation constant (Kd) at pH 7.0 and 25 degrees C, similar to that observed with other flavodoxins. In addition, the apoflavodoxin readily binds riboflavin (Kd = 0.72 microM; 50 mM sodium phosphate, pH 7.0, 5 mM EDTA at 25 degrees C) and the complex is spectroscopically very similar to that formed with FMN. The redox potentials for the riboflavin complex were determined at pH 6.5 (E1 = -262 mV, E2 = -193 mV; 25 degrees C) and are discussed in the light of earlier proposals that charge/charge interactions between different parts of the flavin hydroquinone play a crucial role in determining E1 in flavodoxin.

Amino Acid Sequence↗
PubMed1990

Effects of substituting asparagine for glycine-61 in flavodoxin from Desulfovibrio vulgaris (Hildenborough).

Gly-61 in flavodoxin from Desulfovibrio vulgaris (Hildenborough) has been changed to Asn by site-directed mutagenesis of the cloned gene. Values determined for the dissociation constant for the dissociation of the mutant protein into apoprotein and FMN, and for the redox potentials of the two 1-electron steps in the reduction of the bound flavin showed that FMN in all three redox states is bound more weakly in the mutant protein than in the wild-type flavodoxin. However, the greatest effect was on the semiquinone, for which Kd is 920 times larger in the mutant. The side-chain of Asn-61 in the mutant may hinder a redox-linked conformational change that occurs in this region of the protein, and which is thought to lead to formation of a hydrogen bond between N(5)H of FMNH and the backbone carbonyl group of amino acid-61.

Asparagine↗
PubMed1986

Properties of the complexes of riboflavin 3',5'-bisphosphate and the apoflavodoxins from Megasphaera elsdenii and Desulfovibrio vulgaris.

Megasphaera elsdenii and Desulfovibrio vulgaris apoflavodoxins have been reconstituted with riboflavin 3',5'-bisphosphate. Several biochemical and biophysical properties of the complexes have been investigated and the results are compared with the properties of the native proteins. The dissociation constant of the modified complex of M. elsdenii flavodoxin is increased by a factor of about 23 by comparison with that of the native protein. The rate constant for the formation of the complex of M. elsdenii flavodoxin is about 26 times lower than that for the native protein. The redox potential of the transition between the oxidized and semiquinone state is similar to that of the native protein. On the other hand, the redox potential of the semiquinone-hydroquinone transition is about 20 mV more negative than that of the native protein. Absorbance and circular dichroic spectra of the protein-bound artificial prosthetic group and the protein-bound natural prosthetic group are very similar. In both the oxidized and in the fully reduced state only minor differences in interaction between the isoalloxazine ring and the apoprotein for the two flavin derivatives are found by 13C and 15N NMR. 31P-NMR studies show that the 5'-phosphate group of the two flavin derivatives is bound in the same way and that it is dianionic in the complex. In contrast, the 3'-phosphate group in riboflavin 3',5'-bisphosphate is monoanionic or even neutral when bound to the protein. The 3'-phosphate group is also close to or on the surface of the protein. Desulfovibrio vulgaris apoflavodoxin has an affinity for riboflavin 3',5'-bisphosphate which is 10 times higher as compared to Megasphaera elsdenii apoflavodoxin (Ka = 10(8) M-1). Also the association rate constant of Desulfovibrio vulgaris apoprotein and riboflavin 3'5'-bisphosphate is found to be 10 times faster than for the Megasphaera elsdenii flavodoxin reaction. The dissociation behaviour of native Desulfovibrio vulgaris flavodoxin measured under identical conditions as for the riboflavin 3',5'-bisphosphate analog gives a value (Kd approximately equal to 0.2 nM) which is considerably lower than reported earlier [Dubourdieu, M., MacKnight, M. L. & Tollin, G. (1974) Biochem. Biophys. Res. Commun. 60, 649-655]. The results are discussed in the light of the existing crystallographic data of flavodoxins and the recently proposed theory on the regulation of the redox potential in flavoproteins [Moonen, C. T. W., Vervoort, J. & Müller, F. (1984) in Flavins and flavoproteins, pp. 493-496, Walter de Gruyter, Berlin].

Apoproteins↗
PubMed1986

A comparative carbon-13, nitrogen-15, and phosphorus-31 nuclear magnetic resonance study on the flavodoxins from Clostridium MP, Megasphaera elsdenii, and Azotobacter vinelandii.

The flavodoxins from Megasphaera elsdenii, Clostridium MP, and Azotobacter vinelandii were studied by 13C, 15N, and 31P NMR techniques by using various selectivity enriched oxidized riboflavin 5'-phosphate (FMN) derivatives. It is shown that the pi electron distribution in protein-bound flavin differs from that of free flavin and depends also on the apoflavoprotein used. In the oxidized state Clostridium MP and M. elsdenii flavodoxins are very similar with respect to specific hydrogen bond interaction between FMN and the apoprotein and the electronic structure of flavin. A. vinelandii flavodoxin differs from these flavodoxins in both respects, but it also differs from Desulfovibrio vulgaris flavodoxin. The similarities between A. vinelandii and D. vulgaris flavodoxins are greater than the similarities with the other two flavodoxins. The differences in the pi electron distribution in the FMN of reduced flavodoxins from A. vinelandii and D. vulgaris are even greater, but the hydrogen bond patterns between the reduced flavins and the apoflavodoxins are very similar. In the reduced state all flavodoxins studied contain an ionized prosthetic group and the isoalloxazine ring is in a planar conformation. The results are compared with existing three-dimensional data and discussed with respect to the various possible mesomeric structures in protein-bound FMN. The results are also discussed in light of the proposed hypothesis that specific hydrogen bonding to the protein-bound flavin determines the specific biological activity of a particular flavoprotein.

Azotobacter↗
PubMed1982

A photo-CIDNP study of the active sites of Megasphaera elsdenii and Clostridium MP flavodoxins.

Megasphaera elsdenii and Clostridium MP flavodoxins have been investigated by photo-CIDNP techniques. Using time-resolved spectroscopy and external dyes carrying different charges it was possible to assign unambiguously the resonance lines in the NMR-spectra to tyrosine, tryptophan and methionine residues in the two proteins. The results show that Trp-91 in M.elsdenii and Trp-90 in Cl.MP flavodoxin are strongly immobilized and placed directly above the benzene subnucleus of the prosthetic group. The data further indicate that the active sites of the two flavodoxins are extremely similar.

Binding Sites↗
PubMed1981

An analysis of activity determinations in a series of coupled redox reactions with special reference to hydrogenase.

In this paper a spectrophotometric method is described to study the effects of pH and redox potential on the hydrogen production activity of hydrogenase, which will be applied in the following paper. The theoretical part of this paper gives the mathematical description of a system in which a redox mediator is continuously reduced by a redox system, in a non-rate-limiting way and continuously enzymatically oxidized, in a rate-limiting way. Due to the changes in redox potential during the course of the reaction the ratio reduced/oxidized redox mediator declines gradually. It is possible to relate the changes in this ratio to the actual amount of product formed. The calculation of product formation has been applied to the production of hydrogen by hydrogenase in a system with a redox mediator, whose reduced form functions as the ultimate electron donor for hydrogenase, and the dithionite/(bi)sulphite redox couple as reducing system. The changes in the ratio reduced/oxidized redox mediator as a function of pH and redox potential, followed spectrophotometrically, have been related to the hydrogen production activity of hydrogenase. The second part of the paper deals with the analysis of the theory and describes the conditions under which the method can be used with methyl viologen as redox mediator. The conditions for determining the pH dependence of the hydrogen production activity manometrically are also described.

Dithionite↗