Ralph G. Wilkins

The coordination chemistry of the binuclear iron site in hemerythrin

A. Introduction 195 B. Methemerythrin 197 (i) The Fe(III)Fe(III) site 197 (ii) The reactions 198 (a) Anations 198 (b) Reductions. 199 C. Deoxyhemerythrin 201 (i) The Fe(II)Fe(II)site 201 (ii) The reactions 202 (a) With oxygen. 202 (b) Oxidations 203 (c) Ligand interactions 204 D Semi-methemerythrin 205 (i) The Fe(II)Fe(III)site 205 (ii) The reactions 206 (a) Anations 206 (b) Redox reactions 207 E. Conformationally controlled reactions 208 F. The last ten years 209 Acknowledgements 211 References 211

EPR spectroscopy of semi-methemerythrin

EPR spectra of semi-met forms of octameric hemerythrin from Themiste zostericola, prepared by one electron reduction of methemerythrin or by one electron oxidation of deoxyhemerythrin, have been visualized at liquid helium temperatures. The spectrum of that prepared by one electron reduction has principal g-values of 1.96 +/- 0.01, 1.88 +/- 0.01, and 1.67 +/- 0.02 while that obtained by one electron oxidation has g = 1.95 +/- 0.01, 1.72 +/- 0.01, and 1.68 +/- 0.02. The amplitude of either spectrum decreases with time on incubation at room temperature according to a first order rate with t 1/2 = 5-8 min, apparently because of an intramolecular disproportionation. Similar EPR spectra have been obtained with semi-metmyohemerythrin of T. zostericola and with the octameric semi-met form of Phascolopsis gouldii. However, these forms disproportionate to a much lesser degree. The azide adduct of the octameric semi-met form of T. zostericola has g-values of 1.94 +/- 0.01, 1.85 +/- 0.01, and 1.57 +/- 0.02. Its EPR spectrum differs somewhat from those of the azide adducts of the octamer of P. gouldii and the monomer of T. zostericola although all are resistant to disproportionation. Methemerythrin and deoxyhemerythrin have no EPR spectra even at liquid helium temperature.

Kinetics of interaction of some α- and β-D-monosaccharides with concanavalin A

Abstract The rates of formation and dissociation of concanavalin A with some 4-methylumbelliferyl and p-nitrophenyl derivatives of α- and β- D -mannopyranosides and glucopyranosides were measured by fluorescence and spectral stopped-flow methods. All process examined were uniphasic. The second-order formation rate constants varied only from 6.8 · 104 to 12.8 · 104 M−. s−1, whereas the first-order dissociation rate constants ranged from 4.1. to 220 s−1, all at ph 5.0, I = 0.3 M, and 25°C. Dissociation rates thus controlled the value of binding constant. The effect of temperature on these reactions was examined, from which enthalpies and entropies of activation and of reaction could be calculated. The effects of pH at 25°C on the reaction rates of 4-methylumbelliferyl α- D -mannopyranoside and 4-methylumbelliferyl α- D -glucopyranoside with concanavalin A were examined. The value of the binding constant Kap (derived from the kinetics) at any pH could be related to the intrinsic binding constant K by the expression Kap = KaK(Ka + [H+])−1. The values of Ka, the ionization constant of the protein segment responsive to sugar binding, were 3 · 10−4 M and 1 · 10−4 M for 4-methylumbelliferyl α- D -mannopyranoside and 4-methylumbelliferyl α- D -glucopyranoside, respectively. The binding constant of p-nitrophenyl α- D -mannopyranoside is surprisingly much less sensitive to a pH change from 5.0 to 2.7. Ionic strength had little effect on the binding characteristics of 4-methylumbelliferyl α- D -mannopyranoside to concanavalin A at pH 5.2 and 25°C.

Reduction of methemerythrin by dithionite ion

Abstract The reduction of methemerythrin (Hr + ) by dithionite produces deoxyhemerythrin (Hr o ) in multi, possibly three, stages. The kinetics were examined at pH 8·2 and 25 °C. The first stage is reduction of methemerythrin to an intermediate A by SO 2 - (k = 1.3 × 10 5 m −1 s −1 ). The much slower second and third stages have rates independent of dithionite concentrations. Reaction is completed after about 10 h. The kinetics of reactions of A with N 3 - , H 2 O 2 , and O 2 were examined, as well as the conversion of A to intermediate B (k = 4·4 × 10 −4 s −1 ). It is concluded that A is an (Fe(II)Fe(III)) 8 species, and that in B the unit (Fe(II)Fe(II)) 8 is well developed, judging by its unreactivity towards N 3 − , its reaction with H 2 O 2 , and its reversible uptake of O 2 (85–90% of the final product). There is little effect of adjusting the pH to 6·3 on the rates of the processes examined.

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.

Interaction of cobalt(II) bovine carbonic anhydrase with pyridine-2,6-dicarboxylate ion and other chelating ligands

Abstract The removal of cobalt from cobalt(II) bovine carbonic anhydrase by pyridine-2-carboxylate, pyridine-2,6-dicarboxylate and 5-methyl-1,10-phenanthroline occurs via formation of an intermediate. This is presumed to be a ternary adduct of cobalt(II) enzyme with the ligand. In this, metal-protein bonds are loosened, probably via distortion of the normal geometry, resulting in accelerated breakdown of the adduct to apoprotein, compared with the behavior of the cobalt(II) enzyme alone. With 2-carboxy-1,10-phenanthroline, removal of metal is very rapid but no adduct is observed. Values of stability constants of the adducts and rate constants for their decomposition to apoprotein and their formation from apoprotein and cobalt(II) complex were measured at pH 5.5 and 25°C. Formation and dissociation rate constants for the adduct of cobalt carbonic anhydrase with pyridine-2,6-dicarboxylate could be measured from pH 5 to 7 and 10° to 25°C by stopped flow. Values of thermodynamic parameters for the various reactions agreed well with those estimated from the kinetic data.

Reduction potentials of various hemerythrin oxidation states

Abstract The reduction potentials of the couple methemerythrin/(semi-met) r have been determined spectrally, using 2,6-dichlorophenolindophenol as a redox partner, for monomer and octamer from Themiste zostericola and for octamer protein from Phascolopsis gouldii. The reduction potentials of the couple (semi-met)o/deoxy have been determined spectrally, using Fe(III) cyanocomplexes, for monomer and octamer protein from Themiste zostericola. The values of other potentials, (met/(semi-met)o and (semi-met) r /deoxy), have thus been deduced as well as equilibrium constants for the (semi-met) r α (semi-met)o conformational changes for hemerythrins from Themiste zostericola.

Reduction of methemerythrin-anion adducts by dithionite ion.

The reduction by dithionite ion (in excess) of methemerythrin-anion adducts, Hr+X-, to deoxyhemerythrin, Hr degree, has been examined at 25 degrees and pH 6.3 and 8.2. The results accord with the scheme: S2O42- in equilibrium 2SO2- rapid Hr+X- in equilibrium Hr++X- k-1, k1 Hr++SO2- leads to PRODUCT k2 with X- = Br-, HCO2-, CNO-, and F-, k2[SO2-] greater than k1[X-], and the pseudo first-order rate constant, kobs (= k-1), is independent of [X-] and [S2O42-]. Only with X- = NCS- is k2[SO2-] approximately k1[X-] and kobs = a[S2O42-]1/2 (b[NCS-] + [S2OR2-]1/2)-1. Values at pH 6.3 of k-1 (sec-1) and k1 (M-1 sec-1), obtained by anation and anion displacement reactions, are 2.3 x 10(-3), 1.6 x 10(-2) (Br-); 1.5 x 10(-3), 1.2 x 10(-2) (HCO2-); 1.3 x 10(-4), 0.52 (CNO-) and approximately 2 x 10(-4), 3.3 x 10(-3) (CN-, pH 7.0). Values of k-1 from reduction and displacement methods are in good agreement with each other. The value of k2 (1.6 x 10(5) M-1 sec-1, pH 6.3) in somewhat smaller than that for reduction of the met form of hemoproteins. There is only a small effect of pH on rates. Direct reduction of Hr+CN- does not occur, in contrast with Mb+CN-.

Ligational effects on reduction of myoglobin and horseradish peroxidase by inorganic reagents

Previous studies of the reduction of metmyoglobin and adducts by dithionite have been extended to horseradish peroxidase and its complexes. In addition, the reduction of metmyoglobin, horseradish peroxidase and adducts by a much bulkier reactant, cobalt(II) sepulchrate has been studied. Similar patterns of kinetic behavior were observed, namely, direct reduction of cyanide and imidazole adducts of the iron(III) proteins and indirect (via dissociation) reduction of the fluoride adduct. In the reduction of horseradish ferriperoxidase by cobalt(II) sepulchrate, three steps are observed and the spectral properties of the intermediate(s) and their kinetic behavior delineated. The final product is ferroperoxidase confirmed by spectral properties and its behavior on oxygenation. Reduction of cytochrome c(III) and Hipip by cobalt(II) sepulchrate appears to be a uniphasic reaction and second-order rate constants have been determined.

Acid-assisted anion interaction with deoxyhemerythrin.

The interaction of N3-, CNO- and F- with deoxyhemerythrin was monitored from pH 6 to 9 and 3 degrees C to 25 degrees C at an ionic strength 0.5 M (Na2SO4) by using competition with O2. A proton is involved in forming the adduct and is not lost from the protein even at pH values as high as 9. Thermodynamic and kinetic parameters for the interaction of each anion are obtained. It is considered that the proton is introduced as the acid HX and the corresponding parameters for the reaction of HX with deoxyhemerythrin are calculated. These are compared with those for the O2 reaction with deoxyhemerythrin. The adducts are remarkably inert and have dissociation half-lives ranging from 7 s (N3-) to 70 s (CNO- and F-) at 25 degrees C. This stability is ascribed to protonation of a bridged hydroxy group or even bridge-breakage.