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 (MbO2) sperm whale oxymyoglobin, with all accessible His residues alkylated by sodium bromoacetate (CM‐MbO2), and the mutant sperm whale oxymyoglobin [MbO2(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 MbO2 complexing with redox‐inactive Zn2+, 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.
Abstract-The effects of commercial fi shing with crab pots on the physical condition of the snow crab (Chionoecetes opilio) and southern Tanner crab (C. bairdi) were investigated in the Bering Sea and in Russian waters of the Sea of Okhotsk. In crabs that were subjected to pot hauling, the presence of gas embolism and the deformation of gill lamellae were found in histopathological investigations. Crab vitality, which was characterized subjectively through observation of behavioral responses, depended on not only the number of pot hauls but also the time between hauls. Immediately after repeated pot hauls at short time intervals (≤3 days), we observed a rapid decline in vitality of crabs. When hauling intervals were increased to >3 days, the condition of crabs did not significantly change. After repeated pot hauls, concentration of the respiratory pigment hemocyanin ([Hc]) was often lower in the hemolymph of crabs than in the hemolymph of freshly caught animals. Our research indicated that changes in [Hc] in crabs after repeated pot hauls were caused by the effects of decompression and not by starvation of crabs in pots or exposure of crabs to air. We suggest that the decrease in [Hc]
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.
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).
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