A. Geoffrey Sykes

Reactions of the heterometallic cuboidal clusters Mo3MS4 (M = Co, Ni, Pd, Cu) and Mo3NiSe4 with CO: electron counts and kinetic/thermodynamic studies with M = Ni, Pd

Abstract The aqueous solution reactions of heterometallic cube derivatives of [Mo3S4(H2O)9]4+, written here as [Mo3MS4(H2O)10]4+ (tetrahedral M = Fe, Co, Ni, Pd, Cu) with carbon monoxide are considered. Thermodynamically favourable reactions complete in 330 min are detected with M = Co (Group 9), and Ni and Pd (Group 10) containing cubes having 15 and 16 metal electrons, respectively, but no corresponding reactions are observed with M = Fe, Cu cubes having 14 and 17 metal electrons, respectively. A similar reaction is observed with the Se-containing cube [Mo3NiSe4(H2O)10]4+, and with the higher oxidation state cube [Mo3CuS4(H2O)10]5+ (16 electrons) [Mo3S4(H2O)9]4+ and Cu(CO)+ are obtained as final products. On bubbling N, through the latter CO is removed, and approaching quantitative reformation of [Mo3CuS4(H2O)10]5+ is observed. Greater difficulty is experienced in removing carbon monoxide from the other carbonyl adducts (M = Co, Ni, Pd) using N2.

REACTIVITY OF GALACTOSE OXIDASE

Abstract Recent studies on reactions of the two-equivalent CuII/tyrosyl radical containing enzyme galactose oxidase (GOase) from Fusarium NRRL 2903 are referred to in this report. Two GOaseox active-site acid dissociation pKa values have been determined by UV–vis spectrophotometry, and are 5.7 (coordinated H2O) and 7.0 (protonated Tyr-495). The active enzyme (GOaseox) catalyses the oxidation of the primary alcohols RCH2OH+O2→RCHO+H2O2, where previous studies with five different substrates are extended to include saturation kinetics for d -Galactose and d -Raffinose. Two competing steps, GOaseox+RCH2OH→GOaseredH2+RCHO (k1) and GOaseredH2+O2→GOaseox+H2O2 (k2) are observed. Rate constants k1 are dependent on pH, whereas k2 is independent of pH in the range 5.5–9.0. The k2 behaviour suggests that the two protons required to bring about the O2→H2O2 conversion are provided by the protonated form GOaseredH2. Michaelis–Menten kinetics allow Km (=1/Kbind) and kcat (catalytic turnover) to be determined, where Kbind M−1 values are small for d -Galactose (6.7) and d -Raffinose (14.3), in keeping with the enzyme being extracellular. Pulse radiolysis studies on GOasesemi with CO2 − (which is unable to provide protons) are also described. These indicate that, after initial reduction to the CuI state (GOasered), a spontaneous decay of the unstable product occurs with formation of a disulfide radical anion (RSSR −) detected by its absorbance at 450 nm. Slow decay of the disulfide radical is observed in a further step. Evidence obtained suggests that the spontaneous decay of the tyrosyl radical of GOaseox also involves the disulfide. As a means of modelling substrate binding, NCS− substitution at the CuII active site of GOaseox has been investigated. Dependence on pH is again observed and at 25°C, pH 7.0 (10 mM phosphate), K=0.5×103 M−1, with forward rate constant kf=1.1×104 M−1 s−1, I=0.100 M (NaCl).

Structure, Properties and Reactivity of the FeIIFeIII and ZnIIFeIII Purple Acid Phosphatases

This microreview describes the structure, properties and mechanisms of the purple acid phosphatases (PAP). The enzyme is isolated from mammalian, plant and bacterial sources. X-ray structural information is now available for the enzyme from pig (uteroferrin), rat and kidney beans. Features of the mechanism are the concerted action of a labile MII centre (FeII or ZnII) alongside a more inert FeIII. The latter is effective as a conjugate-base FeOH2+, which initiates hydrolysis at the MII-bound phosphate ester by a process involving OH− replacement of OR− at the PV. Histidine residues near to the active site help bind the phosphate and are involved in the release of OR−. Effects of replacement of the FeII by MnII, CoII, NiII, CuII and ZnII, and of FeIII by GaIII,AlIII and InIII have been studied. The mechanistic role of the ZnIIZnII combination in alkaline phosphatases, and other related dinuclear centres is also considered.

Kinetics of the equilibration of oxygen with monomeric and octameric hemerythrin from Themiste zostericola

Single relaxations for the equilibration of O2 with monomeric and octameric deoxy forms of hemerythrin from Themiste zostericola have been observed at 25°C using the temperature-jump technique. At 25°C, pH 8.2 (Tris/H2SO4 and I = 0.10 M (Na2SO4), formation rate constants kon are 7.8 · 107 M−1 · s−1 and 7.5 · 106 M−1 s−1, respectively. The procedure used does not give a precise measure (small intercepts) of dissociation rate constants, koff. These were determined instead by the stopped-flow method using dithionite to induce dissociation of the oxy protein. Values of koff for the monomer (3.1 · 102 s−1) and octamer (82 s−1), in association with kon values, lead to equilibrium constants for the formation of oxyhemerythrin of 2.5 · 105 M−1 and 0.9 · 105 M−1, respectively, at 25°C, pH 8.2 and I = 0.10 M (Na2SO4). These latter are in reasonable agreement with values (1.5 105 M−1 and 1.3 · 105 M−1) determined spectrally on the equilibrated solutions. Using the octameric protein, it was shown that replacement of SO42− by ClO4− or Cl− ions (at a constant I = 0.10 M) led to an approximately 2-fold enhancement of kon but had little effect on koff. The addition of Ca2+ or Mg2+ ions (0.01 M), with or without 0.50 M NaCl, also gives up to 4-fold increases in kon, but unchanged koff values. Oxygen pulse experiments on the octamer show no effect on koff of the degree of oxygenation of the protein. A comparison is made with similar data for hemoglobin, myoglobin and hemocyanin.

High-yield synthesis of the cuboidal rhenium cluster [Re4S4(CN)12]4− by reaction of the triangular cluster [Re3S7Br6]+ with cyanide

Abstract Reaction of the triangular cluster compound [Re3S7Br6]Br with an aqueous solution of KCN results in the conversion of trinuclear Re3v to cuboidal Re4IV, which is isolated here as Cs2K2[Re4S4(CN)12]·2H2O in 78% yield and characterized crystallographically. The reaction is believed to proceed via trinuclear Re3VS47+ to binuclear Re2VIS44+ with the exclusion of ReIII. The S2− → S and CN− → SCN− changes result in dimerization to the Re4IVS48+ product.

Kinetic studies on the reduction of the tyrosyl radical of the R2 subunit of E. coli ribonucleotide reductase

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.

Kinetic data for redox reactions of cytochrome c with Fe(CN)5X complexes and the question of association prior to electron transfer

Use of rigorous equilibration kinetics to evaluate rate constants for the Fe(CN)6 4- reduction of horse-heart cytochrome c in the oxidized form, cyt c (III), has shown that limiting kinetics do not apply with concentrations of Fe(CN)6 4- (the reactant in excess) in the range 2-10 x 10(-4) M, I = 0.10 M (NaCl). The reaction conforms to a first-order rate law in each reactant, and at 25 degrees C, pH 7.2 (Tris), it is concluded that K for association prior to electron transfer is less than 200 M-1. From previous studies at 25 degrees C, ph 7.0 (10(-1) M phosphate), I = 0.242 M (NaCl), a value K = 2.4 x 10(3) M-1 has been reported. Had such a value applied, some or all of the redox inactive complexes Mo(CN)8 4-, Co(CN)6 3-, Cr(CN)6 3-, Zr(C2O4)4 4- present in amounts 5-20 x 10(-4) M would have been expected to associate at the same site and partially block the redox process. No effect on rats was observed. With the reductants Fe(CN)5(4-NH2-py)3- and Fe(CN)5(imid)3-, reactions proceeded to greater than 90% completion and rate laws were again first order in each reactant. Rate constants (M-1 sec-1) at 25 degrees C, pH 7.2 (Tris), I = 0.10 M (NaCl), are Fe(CN)6 4- (3.5 x 10(4)), Fe(CN)5(4-NH2py)3- (6.7 x 10(5), and Fe(CN)5(imid)3- (4.2 x 10(5). Related reactions in which cyt c(II) is oxidized are also first order in each reactant, Fe(CN)6 3- (9.1 x 10(6)), Fe(CN)5(NCS)3- (1.3 x 10(6)), Fe(CN)5(4-NH2py)2- (3.8 x 10(6) at pH 9.4), and Fe(CN)5(NH3)2- (2.75 x 10(6) at ph 8). Redox inactive Co(CN)6 3- (1.0 x 10(-3) M) has no effect on the reaction of Fe(CN)6 3- which suggests that a recent interpretation for the Fe(CN)6 3- oxidation of cyt c(II), I = 0.07 M, may also require reappraisal.

Kinetics of the rapid monomer–dimer equilibration of molybdenum(VI) in aqueous perchloric acid solutions

The monomer–dimer equilibration of molybdenum(VI) in 0·2–3·0M perchloric acid solutions, I= 3·0M(LiClO4), has been studied at 25 °C. Spectrophotometric measurements are consistent with the presence of a monomeric species, [HMoO3]+, and dimeric species, [H2Mo2O6]2+ and [H3Mo2O6]3+, in agreement with previous work by Krumenacker. Evidence for the further monomeric species [H2MoO3]2+ has been obtained. Kinetic studies using the temperature-jump technique indicate a major pathway (i) for equilibration, with rate constant K1=[HMoO3]++[HMoO3]+ [graphic omitted] [H2Mo2O6]2+(i)(1·71 ± 0·10)× 105 l mol–1s–1 and k–1=(3·20 ± 0·20)× 103s–1. The equilibration(ii), appears to contribute [H2MoO3]2++[HMoO3]+ [graphic omitted] [H3Mo2O6]3+(ii) to a small extent with k2=(0·3 ± 0·3)× 105 l mol–1s–1 and k–2=(30 ± 20) s–1.

Pulse-radiolysis studies on the oxidised form of the multicopper enzyme ascorbate oxidase: evidence for two intramolecular electron-transfer steps

Two intramolecular electron-transfer steps have been identified in pulse-radiolysis studies on the multicopper enzyme ascorbate oxidase, which has four Cu atoms in the catalytically active monomer form. The enzyme was initially in the fully oxidised CuII4 state. Pulse radiolysis was carried out at 19 °C, pH 7.0 (40 mM phosphate), l= 0.100 M, in the first instance with formate to generate CO2˙– as the only (reducing) radical present. When in addition appropriate amounts of methyl viologen (1,1′-dimethyl-4,4′-bipyridinium, dmbipy2+), deazaflavin, or lumiflavin were present the CO2˙– was rapidly converted into CO2 with concomitant formation of the corresponding radical form (e.g. dmbipy˙+) as the only reactive species. Reactions of all four radicals with ascorbate oxidase (reactant in excess) give a metastable type 1 copper reduced product. Contrary to earlier reports two intramolecular electron-transfer steps k1 and k2 follow in which the colour of the type 1 site is restored. Both are independent of the radical type used. Thus the first stage is assigned as electron transfer from the type 1 CuI to the trinuclear combined type 3/type 2 site. Rate constants k1 and k2/s–1 are for CO2˙–(120, 2.0), dmbipy˙+(127, 2.3), deazaflavin (121, 2.5) and lumiflavin (97, 2.4). Mechanistic assignments for the two stages are considered, and an apparent disagreement with a previous study is explained.

Kinetic studies on 1:1 electron transfer reactions involving blue copper proteins: 8. Reactions of plastocyanin and azurin with cytochrome c and high potential iron-sulfur protein

Abstract Rate constants determined by the stopped-flow method for four protein-protein reactions at 25°C, pHs in the range 5.8–7.5. I = 0.10 M (NaCI), are as follows: cytochrome c(II) with plastocyanin, PCu(II). 1.5 × 10 6 M −1 sec −1 , pH 7.6; high-potential iron-sulfur protein (Hipip) with PCu(II), 3.7 × 10 5 M −1 sec −1 . pH 5.8; cytochrome c(II) with azurin, ACu(ll). 6.4 × 10 3 M −1 sec −1 , pH 6.1; Hipip with ACu(II), 2.2 × 10 5 M −1 sec −1 , pH 5.8. Activation parameters have been determined for all four reactions; they indicate higher enthalpy requirements and less negative entropy requirements for the PCu(II) as opposed to ACu(II) reactions. Equilibrium constants K for association prior to electron transfer are −1 for the cytochrome c(II) reduction of PCu(II) (estimated charges 8 + and 9-,respectively), and −1 for the other reactions, indicating no favorable interactions. Rate constants have been analyzed in terms of the simple Marcus theory, which has previously given an excellent fit to thirteen protein-protein reactions considered by Wherland and Pecht. No similar correlation exists in the present studies, and calculated rate constants differ by orders of magnitude from experimentally determined values.