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A.G. Sykes
Biochemical Education. 06/2010; 5(1):4 - 4.
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ChemInform 09/2004; 35(40).
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04/2002;
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04/2002;
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ABSTRACT: The purple corner-shared double cube [Mo(6)HgS(8)(H(2)O)(18)](8+) derivative of green [Mo(3)S(4)(H(2)O)(9)](4+), obtained under air-free conditions by the reaction with Hg(0) (metal), is also formed with Hg(I)(2). The Hg(I)(2) reaction is accounted for by the disproportionation Hg(I)(2) <==> Hg(0) + Hg(II), which is a source of Hg(0). X-ray crystallographic information on the blue partially Cl(-) substituted cucurbituril supramolecular assemblies [Mo(6)HgQ(8)Cl(4)(H(2)O)(14)](C(36)H(36)N(24)O(12))Cl(4).14H(2)O (1) and of the Se analogue [Mo(6)HgSe(8)Cl(4) (H(2)O)(14)](C(36)H(36)N(24)O(12))Cl(4).14H(2)O (2) have been determined. The product [W(6)HgSe(8)Cl(4)(H(2)O)(14)](C(36)H(36)N(24) O(12)) Cl(4).14H(2)O (3) has also been obtained, but there is no evidence for [W(6)HgS(8)(H(2)O)(18)](8+) and related forms. The formation of [Mo(6)HgS(8)(H(2)O)(18)](8+) by the reaction of [Mo(3)S(4) (H(2)O)(9)](4+) with Hg(0) under anaerobic conditions maximizes after approximately 40 h in 2.0 M HCl, but requires longer reaction time ( approximately 120 h) in 2.0 M Hpts (p-toluenesulfonic acid) and in 2 M HClO(4) ( approximately 6 days). In 2.0 M HCl there is little absorbance increase until [Mo(3)S(4)(H(2)O)(9)](4+) exceeds 1.2 x 10(-)(3) M, which is explained by a dependence of the formation K (265 M(-1)) on [Mo(3)S(4)(H(2)O)(9)(4+)](2). Furthermore, on dilution of column-purified [Mo(6)HgS(8)(H(2)O)(18)](8+), Beer's law is not obeyed and equilibria involving 2[Mo(3)S(4)(H(2)O)(9)](4+) are apparent. The kinetics of formation of [Mo(6)HgS(8)(H(2)O)(18)](8+) is first-order in [Mo(3)S(4)(H(2)O)(9)](4+), consistent with rate-determining formation of the single cube [Mo(3)HgS(4)(H(2)O)(x)](4+). The oxidations of [Mo(6)HgS(8)(H(2)O)(18)](8+) with [Fe(H(2)O)(6)](3+) and [Co(dipic)(2)](-) are complicated by the release of [Hg(H(2)O)(6)](2+), which also functions as an oxidant. Similar results are obtained for [Mo(6)HgSe(8)(H(2)O)(18)](8+) and the less extensively studied [W(6)HgSe(8)(H(2)O)(18)](8+).
Inorganic Chemistry 12/2001; 40(26):6598-603. · 4.60 Impact Factor
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ABSTRACT: Studies leading to the incorporation of Group 14 germanium into the incomplete cuboidal clusters [M(3)E(4)(H2O)(9)](4+) (M = Mo, W; E = S, Se) have been carried out. From the clusters [Mo(3)E(4)(H2O)(9)](4+), corner-shared double cubes [Mo(6)GeE(8)(H2O)(18)] are obtained with GeO, by heating with Ge powder at 90 degrees C, or by heating with GeO(2) in the presence of H(3)PO(2) as reductant at 90 degrees C, illustrating the dominance of the double cubes. The yellow-green single cube [Mo(3)GeS(4) (H2O)(12)](6+) is only obtained by controlled air oxidation of [Mo(6)GeS(8)(H2O)(18)](8+) over a period of approximately 4 days followed by Dowex purification. In the case of the trinuclear clusters [W(3)E(4)(H2O)(9)](4+), the single cubes [W(3)GeE(4)(H2O)(12)](6+) are dominant and prepared by the reactions with GeO, or GeO(2)/H(3)PO(2). Conversion of [W(3)GeE(4)(H2O)(12)](6+) to the corresponding double cubes is achieved by reductive addition with BH(4)(-) in the presence of a further equivalent of [W(3)E(4)(H2O)(9)](4+). The crystal structures (pts(-) = p-toluene-sulfonate) of [Mo(6)GeS(8)(H2O)(18)](pts)(8).28H2O, (1); [W(6)GeS(8)(H2O)(18)](pts)(8).23H2O, (2); and [Mo(6)GeSe(8)(H2O)(18)](pts)(8).8H2O, (3); have been determined, of which (2) is the first structure of a W(6) double cube. The M-M bond lengths of approximately 2.7 A are consistent with metal-metal bonding, and the M-Ge of approximately 3.5 A corresponds to nonbonding separations. Of the Group 13-15 corner-shared double cubes from [Mo(3)S(4)(H2O)(9)](4+), [Mo(6)GeS(8)(H2O)(18)](8+) is the least reactive with [Co(dipic)(2)](-) as oxidant (0.077 M(-1) s(-1)), and [Mo(6)SnS(8)(H2O)(18)](8+) is next (14.9 M(-1) s(-1)). Both Ge and Sn (Group 14) have an even number of electrons, resulting in greater stability. In contrast, [W(6)GeS(8)(H2O)(18)](8+) is much more reactive (7.3 x 10(3) M(-1) s(-1)), and also reacts more rapidly with O(2).
Inorganic Chemistry 12/2001; 40(24):6115-22. · 4.60 Impact Factor
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ABSTRACT: A procedure for the preparation of the fully reduced Cu(I) form of galactose oxidase, GOase(red), involving reduction of GOase(semi) (or GOase(ox)) with non-coordinating [Ru(NH(3))(6)](2+) (51 mV vs. nhe) is described. Air-free conditions and a two-fold excess of [Ru(NH(3))(6)](2+) give a stable product with no further UV-Vis changes over >1.5 h. Rate constants for the reduction of GOase(semi) (k(f)=860 M(-1) s(-1)) give a first-order [H(+)]-dependence (pK(1a)=7.9), but the reverse process involving [Ru(NH(3))(6)](3+) oxidation of GOase(red) (k(b)=18.6 M(-1) s(-1)) is independent of pH (5.5 to 9.5). The reduction potential E(2)(o)' (vs. nhe) for the GOase(semi)/GOase(red) (i.e. Cu(II)/Cu(I)) couple is 149 mV at pH 7.5, which varies from 160 mV (pH 5.5) to 120 mV (pH 10.5), suggesting pK(1a) (GOase(semi)) and pK(2a) (GOase(red)) acid dissociation constants both involving Tyr-495. It is concluded that pK(2a) is for acid dissociation of uncoordinated H(+)Tyr-495. Consistent with this interpretation rate constants/M(-1) s(-1) for the GOase(semi) Tyr495 Phe variant, k(f)=1.59x10(3) and k(b)=16.1, respectively, are independent of pH and give a reduction potential of 169 mV. Comparisons are made of reduction potentials (E(1)(o)'/mV pH 7.5) for the GOase(ox)/GOase(semi) (i.e. Tyr(.)/Tyr) couple, and are for the Cys228Gly variant (630), for enzyme with N(3)(-) for H(2)O at the substrate binding exogenous site (393), and for apo-protein (570). These compare with previously reported values for the variants Trp290His (730) and Tyr495Phe (450), and together serve to quantify different contributions to the unusually small E(1)(o)' of 400 mV for the Tyr(.)/Tyr couple. At pH 7.5 the reduction potential for the two-equivalent GOase(ox)/GOase(red) couple is calculated to be 275 mV. The rate constant for the reaction of GOase(red) with GOase(ox) is 4.4x10(3) M(-1) s(-1) at pH 7.5.
Journal of Inorganic Biochemistry 07/2001; 85(4):237-43. · 3.35 Impact Factor
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ABSTRACT: Solutions of galactose oxidase stored in air give in 3-4 h a mix of GOase(ox)(Cu(II)-Tyr(*)) and GOase(semi)(Cu(II)-Tyr), as a result of processes involving the formation and decay of the Cu(II)-coordinated tyrosyl radical (Tyr(*)). In this work the two reactions have been studied by UV-vis spectrophotometry and separate rate laws defined. The first involves the "spontaneous" autoreduction of GOase(ox) to GOase(semi), which in air-free conditions is 100% complete. Rate constants (k(red)) are dependent on pH, and previously defined acid dissociation constants pK(1a) = 5.7 (exogenous H(2)O ligand), and pK(2a) = 8.0 (axial H(+)Tyr-495) apply. Values of k(red)(25 degrees C) range from 1.55 x 10(-4) s(-1) (pH 5.5) to 2.69 x 10(-4) s(-1) (pH 8.6), I = 0.100 M (NaCl). No reaction occurs with N(3)(-) or NCS(-) present in amounts sufficient to give >98% binding at the substrate binding (exogenous) site, while CH(3)CO(2)(-) and phosphate (less extensively bound) also inhibit the reaction. From such inhibition studies K(25 degrees C) is 161 M(-1) at pH 6.4 for acetate (previous value 140 M(-)(1)) and 46 M(-1) at pH 7.0 for phosphate. No reaction occurs when the disulfide Cys515-Cys518 (10.2 A from the Cu) is chemically modified with HSPO(3)(2-), and electron transfer via the disulfide and exogenous position is proposed (source of the electron not established). The conversion of GOase(semi) to GOase(ox) only occurs with O(2) present, when a first-order dependence on [O(2)] is observed, giving k(ox)(25 degrees C) = 0.021 M(-1) s(-1) at pH 7.5. This process is unaffected by NCS(-) or N(3)(-) bound at the exogenous site, and a mechanism involving outer-sphere reaction of O(2) to O(2)(-) followed by a fast step O(2) to H(2)O(2) is proposed. As GOase(ox) is formed, autoreduction back to GOase(semi) occurs, and at pH 7.5 with O(2) in large excess (1.13 mM) the maximum conversion to GOase(ox) is 69%. The k(ox) reaction proceeds to completion with >98% N(3)(-) bound at the exogenous site.
Inorganic Chemistry 05/2001; 40(11):2528-33. · 4.60 Impact Factor
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ABSTRACT: This Account reports recent progress in the study of some approximately 20 heterometal derivatives of [Mo3S4(H2O)9]4+ with reference also to W and Se analogues. Single cubes (3:1) and corner-shared double cubes (6:1), as well as dimers of the 3:1 single cubes, are considered. A classification of the heterometals as subtypes A, B, and C is introduced.
Accounts of Chemical Research 04/2001; 34(3):223-30. · 21.64 Impact Factor
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ABSTRACT: The self-exchange rate constant (25 degrees C) for parsley plastocyanin is 5.0 x 10(4) M-1 s-1 at pH* 7.5 (I = 0.10 M). This value is quite large for a higher plant plastocyanin and can be attributed to a diminished upper acidic patch in this protein. The self-exchange rate constant is almost independent of pH* in the range 7.5-5.6, with a value (25 degrees C) of 5.6 x 10(4) M-1 s-1 at pH* 5.6 (I = 0.10 M). At this pH*, the ligand His87 is protonated in approximately 50% of the reduced protein molecules (pKa* 5.6), and this would be expected to hinder electron transfer between the two oxidation states. However, this effect is counterbalanced by the enhanced association of two parsley plastocyanins at lower pH* due to the partial protonation of the acidic patch.
Inorganic Chemistry 02/2001; 40(2):354-60. · 4.60 Impact Factor
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ABSTRACT: Thermodynamic and kinetic studies on the X- = NCS-, N3-, and CH3CO2- replacement of H2O/OH- at the CuII exogenous site of the tyrosyl-radical-containing enzyme galactose oxidase (GOaseox) from Fusarium (NRR 2903), have been studied by methods involving UV-vis spectrophotometry (25 degrees C), pH range 5.5-8.7, I = 0.100 M (NaCl). In the case of N3- and CH3CO2- previous X-ray structures have confirmed coordination at the exogenous H2O/OH- site. From the effect of pH on the UV-vis spectrum of GOaseox under buffer-free conditions, acid dissociation constants of 5.7 (pK1a; coordinated H2O) and 7.0 (pK2a; H+Tyr-495) have been determined. At pH 7.0 formation constants K(25 degrees C)/M-1 are NCS- (480), N3- (1.98 x 10(4)), and CH3CO2- (104), and from the variations in K with pH the same two pKa values are seen to apply. No pK1a is observed when X- is coordinated. From equilibration stopped-flow studies rate constants at pH 7.0 for the formation reaction kf(25 degrees C)/M-1 s-1 are NCS- (1.13 x 10(4)) and N3- (5.2 x 10(5)). Both K and kf decrease with increasing pH, consistent with the electrostatic effect of replacing H2O by OH-. In the case of the GOaseox Tyr495Phe variant pK1a is again 5.7, but no pK2a is observed, confirming the latter as acid dissociation of protonated Tyr-495. At pH 7.0, K for the reaction of four-coordinate GOaseox Tyr495Phe with NCS- (1.02 x 10(5) M-1) is more favorable than the value for GOaseox. Effects of H+Tyr-495 deprotonation on K are smaller than those for the H2O/OH- change. The pK1a for GOasesemi is very similar (5.6) to that for GOaseox (both at CuII), but pK2a is 8.0. At pH 7.0 values of K for GOasesemi are NCS- (270 M-1), N3- (4.9 x 10(3)), and CH3CO2- (107).
Inorganic Chemistry 02/2001; 40(2):294-300. · 4.60 Impact Factor
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ABSTRACT: The recently reported NMR solution structure of FeIIIFeIII parsley FdI has made possible 2D NOESY NMR studies to determine the point of attachment of CrIIIL in FeIIIFeIII...CrIIIL. The latter Cr-modified product was obtained by reduction of FeIIIFeIII parsley and spinach FdI forms with [Cr(15-aneN4) (H2O)2]2+ (15-aneN4 = 1,4,8,12-tetraazacyclopentadecane), referred to here as CrIIL, followed by air oxidation and chromatographic purification. From a comparison of NMR cross-peak intensities of native and Cr-modified proteins, two surface sites designated A and B, giving large paramagnetic CrIIIL broadening of a number of amino acid peaks, have been identified. The effects at site A (residues 19-22, 27, and 30) are greater than those at site B (residues 92-94 and 96), which is on the opposite side of the protein. From metal (ICP-AES) and electrospray ionization mass spectrometry (EIMS) analyses on the Cr-modified protein, attachment of a single CrIIIL only is confirmed for both parsley and spinach FdI and FdII proteins. Electrostatic interaction of the 3+ CrIIIL center covalently attached to one protein molecule (charge approximately -18) with a second (like) molecule provides an explanation for the involvement of two regions. Thus for 3-4 mM FeIIIFeIII...CrIIIL solutions used in NMR studies (CrIIIL attached at A), broadening effects due to electrostatic interactions at B on a second molecule are observed. Experiments with the Cys18Ala spinach FdI variant have confirmed that the previously suggested Cys-18 at site A is not the site of CrIIIL attachment. Line broadening at Val-22 of A gives the largest effect, and CrIIIL attachment at one or more adjacent (conserved) acidic residues in this region is indicated. The ability of CrIIL to bind in some (parsley and spinach) but not all cases (Anabaena variabilis) suggests that intramolecular H-bonding of acidic residues at A is relevant. The parsley and spinach FeIIFeIII...CrIIIL products undergo a second stage of reduction with the formation of FeIIFeII...CrIIIL. However, the spinach Glu92Ala (site B) variant undergoes only the first stage of reduction, and it appears that Glu-92 is required for the second stage of reduction to occur. A sample of CrIIIL-modified parsley FeIIIFeIII Fd is fully active as an electron carrier in the NADPH-cytochrome c reductase reaction catalyzed by ferredoxin-NADP+ reductase.
Inorganic Chemistry 05/2000; 39(8):1755-64. · 4.60 Impact Factor
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ABSTRACT: Further to a linear free-energy correlation of cross-reaction rate constants k12 for the reaction of eight organic radicals (OR), e.g. MV*+, from methyl viologen, with cytochrome c(III), we consider here similar studies for the reduction of the R2 protein of Escherichia coli ribonucleotide reductase, which has FeIII2 and Tyr* redox components. The same two techniques of pulse radiolysis and stopped-flow were used. Cross-reaction rate constants (22 degrees C) at pH 7.0, I=0.100 M (NaCl), were determined for the reduction of active-R2 with the eight ORs, reduction potentials E0(1) from -0.446 to +0.194 V. Samples of active-R2 have an FeIII2 met-R2 component, which in the present studies was close to 40%. Concurrent reactions have to be taken into account for the five most reactive ORs, corresponding to reduction of the FeIII2 of met-R2 and then of active-R2. Separate experiments on met-R2 reproduced the first of these rate constants, which on average is approximately 66% larger than the second rate constant. A single Marcus free-energy plot of log k12-0.5 log10f versus -E0(1)/0.059 describes all the data and the slope of 0.54 is in satisfactory agreement with the theoretical value of 0.50. Such behaviour is unexpected since the Tyr* is a much stronger oxidant (E0 approximately 1.0 V versus NHE) as compared to FeIII2 (E0 close to zero). X-ray structures of the met- and red-R2 states have indicated that electroneutrality of the approximately 10 A buried active site is maintained. Proton transfer is therefore proposed as a rapid sequel to electron transfer. Other reactions considered are the much slower conventional time-range reductions of active-R2 with hydrazine and dithionite. For these reactions one and/or two-equivalent changes are possible. With both reductants, met-R2 reacts about four-fold faster than active-R2, and as with the ORs the less strongly oxidising FeIII2 component is reduced before the Tyr*.
Journal of Inorganic Biochemistry 05/2000; 79(1-4):59-65. · 3.35 Impact Factor
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ABSTRACT: The [2Fe-2S]ferredoxin I (Fd I) from parsley leaves (Mr = 10,500; 96 amino acids) in the Fe(III)-Fe(III) oxidized form has been studied by 1H-NMR spectroscopy. Sequence-specific 1H-NMR assignments were obtained through two-dimensional classical double-quantum-filtered-COSY, NOESY and TOCSY spectra. NOEs between protons as close as 5.6 A from the paramagnetic Fe(III) atoms were observed at 800 MHz. A total of 3066 NOEs (of which 2533 are meaningful) and 18 distance constraints taken from X-ray crystallography of the Fe2S2 active site were used to obtain the solution structure. From inversion recovery NOESY experiments, 33 longitudinal relaxation rate (Qpara) constraints were used for the structural refinement. The final structure was obtained by a process of restrained energy minimization. Root-mean-square (rmsd) deviation values obtained for the family of 18 structures (with reference to the average structure) are 0.52 +/- 0.10 A and 0.91 +/- 0.12 A for backbone and all heavy atoms respectively. The structure consists of seven-strands of beta-sheets and four short alpha-helices. The quality of the present solution structure is among the best of those reported for [2Fe-2S]ferredoxins. The secondary structure and overall folding are compared with those of Anabaena variabilis Fd and the higher plant Equistum arvense (horse tail) protein determined through X-ray crystallography. The groups believed to be responsible for electron transfer have been analysed.
European Journal of Biochemistry 01/1999; 258(2):465-77. · 3.58 Impact Factor
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ABSTRACT: The detailed mechanism used by [4Fe-4S] ferredoxins to exchange electrons is not known. The importance of two highly conserved aromatic residues, each located close to one cluster of 2[4Fe-4S] ferredoxins has been probed by site-directed mutagenesis of Clostridium pasteurianum ferredoxin. All generated variants are less stable than the native protein and only hydrophobic residues can replace one of the two conserved aromatic residues. With leucine substituting both aromatics, Clostridium pasteurianum ferredoxin cannot even be completely purified because of its deleterious instability. The reduction potentials of Clostridium pasteurianum ferredoxin variants do not depend on the presence of aromatic residues near the clusters. However, the ferredoxin from Entamoeba histolytica which is naturally devoid of aromatic residues displays a reduction potential nearly 60 mV less negative than that of Clostridium pasteurianum ferredoxin. The rate constants for the oxidation of the reduced ferredoxins by the inorganic complexes hexaamine-cobalt(III) chloride and sodium ethylenediaminetetra-acetatecobaltate(III) are similar. This implies that electron transfer from the clusters of these molecules is not mediated by the conserved aromatic residues. These residues rather appear to be involved in maintaining the overall stability of ferredoxins.
Biochimica et Biophysica Acta 08/1996; 1295(2):201-8. · 4.66 Impact Factor
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ABSTRACT: Reduction potentials Eo' for the T. versutus amicyanin couple, AmCuII/I, were determined at pH values in the range 4.4-9.0 by direct measurement using cyclic voltammetry, and from rate constants for the reactions AmCu1 + [Co(terpy)2]3+ and [Co(terpy)2]2+ + AmCuII, using an Eo' for the [Co(terpy)2]2+/3+ couple of 260 mV. At pH > 7.5 the value obtained is 236 mV, which increases with decreasing pH in keeping with proton inactivation of AmCuI. Together with previously determined Eo' values for the T. versutus cytochrome C550 FeIII/FeII couple, it is concluded that the physiologically relevant reaction AmCuI + cyt C550FeIII (kf) is thermodynamically favourable at pH > 6.25, but that the back reaction cyt C550FeII + AmCuII (kb) is favourable at pH < 6.25. Values of kf (25 degrees C) at pH > 6.25 were determined directly by the stopped-flow method, I = 0.100 M (NaCl). At pH < 6.25 kf values were obtained indirectly from the measured kb and equilibrium constants from delta Eo'. The combined kf variations with pH give an acid dissociation pKa for AmCuIH+ of 6.6. In further studies (25 degrees C) rate constants/M-1 S-1 (pH 6.0-8.6) were determined for the cross-reactions of AmCuI with P. aeruginosa azurin AzCuII, and AmCuI with P. aeruginosa cyt C550FeIII, and are 11.0 x 10(5) and 6.4 x 10(5) M-1 S-1 respectively at pH 8.6. Using the Marcus equations corresponding electron self-exchange rate constants (kese/M-1 S-1) of 1.3 x 10(5) and 0.6 x 10(5) M-1 S-1 were calculated for the exchange of AmCuII with unprotonated AmCuI, in good agreement with the value 1.2 x 10(5) M-1 S-1 determined by NMR at pH 8.6. Information was also obtained as to the effect of pH on these kese values.
Biochimica et Biophysica Acta 08/1996; 1295(2):245-52. · 4.66 Impact Factor
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ABSTRACT: Kinetic studies at 25 degrees C, I = 0.100 M (NaCl), on the reduction of the tyrosyl radical of the R2 protein of E. coli ribonucleotide reductase with hydroxyurea (HU), N-methylhydroxylamine, catechol, and seven hydroxamic acid derivatives are reported. There are no pH-dependences in the range 6.2-8.6 investigated except that introduced with N-methylhydroxylamine which itself protonates in this range. At pH 7.6 the rate constant (0.46 M-1 s-1) for the HU reaction is in agreement with earlier values. Slower reactions are observed with the bulkier acetohydroxamic (0.020 M-1 s-1) and benzohydroxamic acids (0.040 M-1 s-1). In the case of N-methylhydroxylamine the rate constant (0.41 M-1 s-1 at pH 7.6) decreases with pH, and it is concluded that the protonated form CH3NH2+OH(pKa = 6.2) has little or no reactivity with Tyr. For this reaction under air-free conditions a second-stage (0.027 M-1 s-1) corresponding to reduction of Fe(III)2 is observed. Mid-point redox potentials for the reductants and estimates of reduction potentials applying in the case of the protein are considered. The reactions with 1,2-dihydroxybenzene (catechol) and 3,4-dihydroxybenzohydroxamic acid (Didox) also have two stages, when the initial Tyr reduction, rate constants/M-1 s-1 for catechol (3.2) and Didox (0.010), is followed by removal of the Fe(III) to give catechol and catechol like Fe(III)-complexed products. The single stage reactions of the hydroxamic acid derivatives which incorporate charged amino-acid groups L-glutamic acid, L-histidine, L-glycine and L-lysine, are slow, and saturation kinetics are observed consistent with association (small K values) prior to redox. The mechanism of reduction of R2-Tyr by all of the reagents studied is discussed.
Biochimica et Biophysica Acta 04/1995; 1247(2):215-24. · 4.66 Impact Factor
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ABSTRACT: Five categories of reaction of the Escherichia coli R2 protein of ribonucleotide reductase (RNR) are defined from mechanistic studies with hydroxyurea, methylhydroxylamine, hydroxamic acid derivatives, long-lived organic radicals of which methyl viologen MV+ is a good example, hydrazine, catechol and their derivatives. Attention is focused on whether a particular reagent reduces only the tyrosyl radical (Tyr.) giving metR2, or the Tyr. and Fe(III)2 in consecutive steps to give fully reduced R2. In the case of hydrazine (N2H4), reduction of both the Tyr and Fe(III)2 occurs in a uniphasic process, while with di-imide (N2H2) it has already been demonstrated that the Tyr. is reduced and that Fe(II)Fe(III) semi-metR2 is formed. A further mechanism is observed with catechol and catechol-like derivatives (in this work Didox), in which there is reduction of the Tyr. in the first stage, followed by scavenging of the Fe(III) in the second. The latter offers a more permanent inactivation of R2, meriting more extensive study in the context of cancer drug therapy. Comparative studies on mouse R2 suggest that the Tyr. and Fe(III)2 of mammalian R2 forms may be more exposed, and as compared to E. coli R2 are approximately an order of magnitude more reactive.
Anti-cancer drug design 03/1994; 9(1):41-50. · 2.38 Impact Factor
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ABSTRACT: The preparation and characterisation of a Ru-modified derivative of the [2Fe-2S] ferredoxin FdI component of A. variabilis by attachment of Ru(NH3)5 to a surface histidine has been carried out. Metal analyses by ICP gave an Fe/Ru ratio of 1.97:1. From NMR the attachment is confirmed as a modification at His-16 and not His-92. No Ru-modification of the [2Fe-2S] FdI component of spinach, which has His-92 but no His-16, was observed. Histidine pKa values have been determined and anomalously low values for A. variabilis (5.3) and spinach (< 5.0) His-92 residues are noted, whereas His-16 gives a pKa of 6.95. Pulse radiolysis techniques with e-aq as reductant have been used to generate the metastable Fe(II)Fe(III)Ru(III) state from Fe(III)2Ru(III), k = 8.8 x 10(10) M-1 s-1. Intermolecular, k(inter) = 4.3 x 10(6) M-1 s-1 at approx. 19 degrees C, but no intramolecular electron-transfer (ET) Fe(II)Fe(III) to Ru(III) contribution was observed with Fe(III)2Ru(III) in the range 7-30 microM. The driving force for ET is approx. 500 mV, and the edge to edge distance from the C gamma of His-16 to the nearest cysteinyl S-atom attached to the Fe2S2 cluster is 16.1 A. Using the Beratan-Onuchic pathway approach, the most favourable ET intramolecular route consists of 20 covalent and 3 H-bonds, with a total distance from the S of Cys-49 to the C gamma of His-16 of 37.1 A. We note that the reduced Fe is on the remote side of the [2Fe-2S] cluster from the site of Ru-attachment.
Biochimica et Biophysica Acta 10/1993; 1202(1):113-20. · 4.66 Impact Factor
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ABSTRACT: The kinetics of the H2O2 oxidation of separated a, b and c subunits (75 kDa, 657 amino acids) of the arthropod Panulirus interruptus hemocyanin (Hc), in the deoxy Cu(I)2 state, have been studied at 25 degrees C, I = 0.100 M (NaCl). Solutions of oxyHc provide small equilibrium amounts of the deoxy reactant deoxyHc + O2<-->oxyHc (K approx. 10(5) M-1). The reaction is of interest because H2O2 is one of the few molecules which is able to access the active site and oxidise deoxyHc to the metHc Cu(II)2 state. The reaction was studied at pH values in the range 6.8-9.6. Traces of Ca2+ (and other 2+ ions) are controlled by addition of EDTA (5 mM). With or without EDTA hexamer forms are present at the lower pH values. At pH > 8.3 and with EDTA added, a and b (3% sequence difference) give monomer forms. The hexamers are however retained at the higher pH values if Ca2+ (10 mM) is added. As in the corresponding studies of deoxyHc with O2, rate constants for subunit c (42% sequence differences) show no variation with pH and the hexamer is retained over the whole pH-range explored. Rate constants for the reactions of H2O2 with the different monomer and hexamer deoxyHc forms of a, b, and c are in the range 1-75 M-1 s-1. A mild pseudo-catalase activity of metHc leading to reformation of oxyHc also contributes to the reaction.
Biochimica et Biophysica Acta 04/1993; 1163(1):17-25. · 4.66 Impact Factor
