The main role of inner histidines in the molecular mechanism of myoglobin oxidation catalyzed by copper compounds.

In the presence of Cu(2+) and Cu(Gly)(2), the oxidation of two native MbO(2)'s (Mb = myoglobin), from the sperm whale and horse, and also two chemically modified sperm whale MbO(2)'s alkylated at solvent-accessible histidines by sodium bromoacetate (CM-MbO(2)) and by iodoacetamide (CA-MbO(2)) have been studied at different pH's, ionic strengths, and concentrations of the copper reagent. The influence of competitive redox-inactive zinc ions on the reaction rate is investigated as well. Localization of Cu(Gly)(2) in sperm whale met-Mb and CM-met-Mb has been examined using the high-resolution NMR method. The obtained data suggest that binding of copper compounds to the surface histidines (all of them are 1.8-2.7 nm apart from the heme) has only a minor, no more than 35%, contribution to the overall reaction rate, in particular under a large excess of the reagent (more than 8-10-fold). The noticeable contribution of His113(116), His48, and His81, which have the greatest affinity to copper according to NMR data, is revealed only at small concentrations of copper, less than a 5-fold excess relative to the protein. The main contribution to the reaction rate must be from the binding of copper to the inner histidines, His97 (0.62 nm from the heme), and possibly to the distal His64. Both are inaccessible to the modification by alkylating reagents and have much lower affinity to copper than all surface histidines, because they are hydrogen-bonded, the former with the carboxyl group of the heme propionate and the second with the liganded O(2).

Ferrocyanide - a novel catalyst for oxymyoglobin oxidation by molecular oxygen.

A comparative study of the rates of ferrocyanide-catalyzed oxidation of several oxymyoglobins by molecular oxygen is reported. Oxidation of the native oxymyoglobins from sperm whale, horse and pig, as well as the chemically modified (MbO(2)) sperm whale oxymyoglobin, with all accessible His residues alkylated by sodium bromoacetate (CM-MbO(2)), and the mutant sperm whale oxymyoglobin [MbO(2)(His119-->Asp)], was studied. The effect of pH, ionic strength and the concentration of anionic catalyst ferrocyanide, [Fe(CN)(6)](4-), on the oxidation rate is investigated, as well as the effect of MbO(2) complexing with redox-inactive Zn(2+), which forms the stable chelate complex with functional groups of His119, Lys16 and Asp122, all located nearby. The catalytic mechanism was demonstrated to involve specific [Fe(CN)(6)](4-) binding to the protein in the His119 region, which agrees with a high local positive electrostatic potential and the presence of a cavity large enough to accommodate [Fe(CN)(6)](4-) in that region. The protonation of the nearby His113 and especially His116 plays a very important role in the catalysis, accelerating the oxidation rate of bound [Fe(CN)(6)](4-) by dissolved oxygen. The simultaneous occurrence of both these factors (i.e. specific binding of [Fe(CN)(6)](4-) to the protein and its fast reoxidation by oxygen) is necessary for the efficient ferrocyanide-catalyzed oxidation of oxymyoglobin.

Mechanism of oxidation of oxymyoglobin by copper ions: comparison of sperm whale, horse, and pig myoglobins.

The influence of Cu2+ concentration, pH, and ionic strength of the solution as well as redox-inactive zinc ions on the rate of oxidation of sperm whale, horse, and pig oxymyoglobins (oxy-Mb) by copper ions has been studied. These myoglobins have homologous spatial structures and equal redox potentials but differ in the number of histidines located on the surface of the proteins. It was shown that oxy-Mb can be oxidized in the presence of Cu2+ through two distinct pathways depending on which histidine binds the reagent and how stable the complex is. A slow pH-dependent catalytic process is observed in the presence of equimolar Cu2+ concentration for sperm whale and horse oxymyoglobins. The curves of pH dependence in both cases are sigmoid with pK(eff) corresponding to the ionization. The process is caused by the strong binding of Cu2+ to His113 and His116, an analogous His residue being absent in pig Mb. In contrast, rapid oxidation of 10-15% of pig oxy-Mb is observed under the same conditions (fast phase), which is not accompanied by catalysis because the reduced copper is apparently not reoxidized. The complexing of Cu2+ with His97 situated near the heme is probably responsible for the fast phase of the reaction. The affinity of His97 for Cu2+ must be significantly lower than those of the "catalytic" His residues since the fast phase does not contribute markedly to the rate of sperm whale and horse oxy-Mb oxidation. Increasing copper concentration does not produce a proportional growth in the oxidation rate of sperm whale and horse oxy-Mbs. Which Cu2+ binding sites of Mb make main contributions to the His reaction rate at different Cu2+/Mb ratios from 0.25 to 10 is discussed.

[Catalytic effect of ferricyanide on the rate of electron transfer between myoglobin and cytochrome c]. / Kataliticheskii éffekt ferritsianida na skorost' perenosa élektrona mezhdu mioglobinom i tsitokhromom c.

The influence of small amounts of low-molecular electron acceptor, potassium ferricyanide, 1 to 20% relative to the cytohrome c concentration, on the rate of electron transfer in the sperm whale oxymyoglobin--horse heart cytochrome c and deoxymyoglobin--cytochrome c systems (under aerobic and anaerobic conditions, respectively) was studied. At low ionic strength, the redox reaction rate was found to increase proportionally to the concentration of ferricyanide in both redox systems. The effect depends on pH in the pH range 5-8, increasing sharply at pH < 6. It was shown that the enhancing of electron transfer is caused by the complexing of [Fe(CN)6]3- with cytohrome c in the Lys72 region, where one of the two strong binding sites for this anion is determined by NMR. Both the high ionic strength and the chemical modification of Lys72 residue inhibit this effect at low ionic strength, markedly decreasing the rate of reaction with myoglobin. Under the same conditions, the effect of ferricyanide in the reaction of oxy-Mb with yeast cytohrome c, which is isopotential to animal cytochromes c but possesses trimethylated Lys72, was several times smaller. In turn, the chemical modification of His residues in myoglobin and the complexing of zinc ion to His119(GH1) almost completely inhibit electron transfer in the systems. Thus, electron transfer between the proteins must proceed through the formation of the Mb.[Fe(CN)6]3-.Cyt c ternary complex, the contacting sites being localized in the His119(GH1) region of myoglobin and near Lys72 of cytohrome c. The increased electron transfer rate in the presence of [Fe(CN)6]3- can be explained by that its binding near Lys72, firstly, provides better electrostatic interactions in the electron transfer complex and, besides, decreases significantly (about 2-fold) the tunneling distance between the two hemes (two lengths of 1.7 and 1.2 nm instead of one of 2.9 nm).

[Oxidation of sperm whale oxymyoglobin, catalyzed by ferrocyanide ions: kinetics and mechanisms]. / Okislenie oksimioglobina kashalota, kataliziruemoe ionami ferrotsianida: kinetika i mekhanizm.

Specific catalytic oxidation of sperm whale oxymyoglobin by small amounts of potassium ferri- and ferrocyanide, from 1 to 20% in relation to the protein concentration, was studied. The mechanism of catalysis was shown to involve specific binding of the ferrocyanide anion to the protein. The influence of pH and ionic strength of the medium, the [Fe(CN)6]4- concentration and of chemical modification of Mb histidines by bromoacetate, as well as the effect of the Mb complexing with redox-inactive zinc ion on the rate of reaction was examined. The zinc ion forms a stable complex with His 119(GH1) on the Mb surface at the equimolar Zn2+ concentration. The kinetic scheme of the reaction was analyzed, and the equilibrium and kinetic parameters were obtained. It was first shown that the strong oxidant such as potassium ferricyanide is able to react with the same protein by two distinct mechanisms: (i) a simple outer sphere electron transfer over the heme edge and (ii) electron transfer after the specific binding of [Fe(CN)6]4- to oxyMb in the His 119(GH1) region, thus catalyzing the protein oxidation.