Oxidation of sperm whale oxymyoglobin, catalyzed by ferrocyanide ions: kinetics and mechanisms

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.     

Study of electron transport in heme proteins. X. Effect of pH, ionic strength, and zinc ions and the rate of ferricytochrome c reduction by oxymyoglobin from swine heart]

The rate of the redox reaction between porcine MbO2 and ferri-Cyt c at different ionic strengths in the pH range 5-8 has been studied. At low ionic strength (I = 0-0.1) the pH dependence curve was found to have a sigmoid shape with pKeff approximately 5.7, implying the effect of ionization of His-119(GH1) at the "active site" of myoglobin on the kinetics of the process. In this range of ionic strengths the rate of the reaction decreases sharply. The slope of the curve in the coordinates of IgKexp versus square root of I/1 + square root of I varies depending on pH. It is greater at pH less than or equal to 6 and smaller at pH 7.5, which is due to deprotonation of His(GH1). At high ionic strength (I greater than 0.1) the rate of electron transfer is negligible, independent of pH and does not practically change as I increases from 0.1 to 1. It is shown that the local electrostatic interactions play a decisive role in the formation of an efficient electron-transfer complex between Mb and Cyt c. The binding of the zinc ion to His(GH1) was found to inhibit the electron transfer at I = 0.01, similarly to what occurs at a high ionic strength, though the "reactive" charges of the proteins are not screened and the positive charge at His(GH1) is retained. This suggests that His(GH1) is directly involved in the mechanism of electron transfer from Mb to Cyt c. The data obtained are compared with earlier data on the effect of pH, ionic strength and zinc ions on the reaction between MbO2 from sperm whale and Cyt c. To explain the higher efficiency of pig MbO2 as electron donor, the electrostatic and steric properties of both myoglobins have been analyzed.     

The oxidation of sperm whale, horse, and pig oxymyoglobins, catalyzed by ferrocyanide ions: kinetics and mechanism

The influence of pH, ionic strength of the solution, and [Fe(CN)6]4- concentration on the rate of oxidation of sperm whale, horse, and pig oxymyoglobins, which is catalyzed by ferrocyanide ions, was studied. These myoglobins have homologous spatial structures and identical redox potentials but differ by the amount of His residues located on the protein surface. The effect of the MbO2 complexing with redox-inactive Zn2+ ion on the reaction rate was also examined. At the equimolar Zn2+ concentration, zinc ions form a stable complex with His119(GH1). It was found that the kinetic behavior of horse MbO2, which lacks His12(A10) substituted for by Gln, is fully analogous to one of sperm whale MbO2, while the oxidation of pig MbO2, three histidines of which, His12, His113(G14), and His116(G17), are replaced by Gln, is strongly inhibited. The mechanism of the catalysis was shown to involve specific binding of [Fe(CN)6]4- to the protein at the His119(GH1) site, which is in accord with the large positive electrostatic potential of this site and the presence here of a cavity large enough to accommodate [Fe(CN)6]4-. The nearby His113 and His116 residiues, which are absent in pig Mb, also play a very important role in the catalysis, because their protonation (especially of the last residue) is most likely responsible for the week oxidation of bound [Fe(CN)6]4- by dissolved oxygen.     

The mechanism of oxymyoglobin oxidation catalyzed by ferrocyanide ions: chemically modified and mutant sperm whale myoglobins

A comparative study of the rate of ferrocyanide-catalyzed oxidation of native sperm whale MbO2, its chemically modified derivative in which all accessible His residues are alkylated by sodium bromoacetate, (CM-MbO2), and mutant sperm whale MbO2 with His119 replaced by Asp residiue, [MbO2(His119-->Asp)] was carried out. The influence of pH, ionic strength, and [Fe(CN)6]4- concentration on the oxidation rate was investigated, as well as the effect of complexing MbO2 with redox-inactive Zn2+ ion, which, at the equimolar Zn2+ concentration, forms a stable complex with His119(GH1) on the protein surface. It was shown that the mechanism of the catalysis involves specific binding of [Fe(CN)6]4- to the protein at the His119(GH1) region, which is in agreement with a large positive electrostatic potential and the presence at this site of Mb of a cavity large enough to accommodate [Fe(CN)6]4- anion. The protonation of nearby His113 and His116 residiues (especially of the latter) plays a very important role in the catalysis, promoting the fast oxidation of bound [Fe(CN)6]4- by dissolved oxygen. Only the presence of these both necessary conditions in MbO2 structure provides its effective oxydation catalyzed by ferrocyanide.     

Electron transport in hemoproteins. IX. The effect of zinc ions on the rate of oxymyoglobin oxidation by ferricytochrome c

The effect of zink ions, which according to the X-ray data are bound to the His GH1 residue of myoglobin, has been investigated. It is shown that the electron transfer in the system is almost completely inhibited at the equimolar Zn2+ concentration in the pH range 5 to 8. Unlike the reaction between the intact MbO2 and Cyt c, the electron transfer rate in this case does not depend on pH and ionic strength of the solution. Further increase of Zn2+ concentration up to the 20-fold molar excess has no significant effect on the rate of the process. Since the thermodynamic characteristics of the redox reaction between MbO2 and Cyt c are not altered in the presence of Zn2+, the findings obtained can be interpreted as indicating the important role of His GH1 in the formation of productive electron transfer complex.     

Controlling the electron transfer rate between oxymyoglobin and ferricytochrome C by alterations in the myoglobin contact site. The role of histidines and electrostatic and steric interactions in the mechanism of the reaction

The kinetics of the redox reaction of sperm whale and pig oxymyoglobins (MbO₂) with ferricytochrome C (CytC) from pig heart has been studied in the pH range 5–8. Also, the effects of histidine (His) modification and of the complexing of both myoglobins with Zn²⁺, on the electron transfer rate, has been investigated. It has been shown that pig MbO₂ reduces Cyt C much more effectively than sperm whale MbO₂. The pH dependence of the reaction rate is shown to result from the influence of two histidines, His 12(A10) and His 119(GH1), in the case of sperm whale myoglobin and only of His GH1 in the case of pig MbO₂. The protonation of His A10 at pH<7.5 decreases the rate of the reaction with Cyt C whereas the ionization of His GH1, on the contrary, increases the electron transfer rate 10–30 times (atI=0.03). The His residues of Cyt C are shown to have no effect on the reaction. Complexing of His GH1 with a zinc ion strongly inhibits the reaction of both sperm whale and pig MbO₂ with Cyt C. The reaction of the zinc-MbO₂ complexes, as distinct from the intact oxymyoglobins, becomes independent of pH and ionic strength. Unlike His A10, His GH1 plays a very important role in the formation of the electron transfer complexes, and is probably directly involved in the charge transfer step. Based on the data obtained, the reactive site of the Mb surface has been identified in the A-GH region. The spatial arrangement of the charged groups in the reactive sites of the two myoglobins has been obtained. The solvent accessibilities of all amino acid residues situated there have been calculated, according to Lee and Richards. In order to explain the different reactivities of sperm whale and pig myoglobins, their electrostatic properties and the steric features of the contact sites have been compared.     

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.     

Oxidation of residue 45 mutant forms of pig deoxymyoglobin with [Fe(CN)6]3-.

The effect of replacement of the highly conserved Lys45 residue in pig myoglobin (Mb) with His, Ser, Glu, and Arg has been investigated. Rate constants/M-1 s-1 at 25 degrees C and pH 8.0, I = 0.100 M (NaCl), for the oxidation of deoxyMb with [Fe(CN)6]3- have been determined, and are for wild-type Lys45 (2.83 x 10(6)), His45 (1.02 x 10(6)), Ser45 (1.12 x 10(6)), Glu45 (0.87 x 10(6)), and Arg45 (3.06 x 10(6)). It is concluded that charge on the residue at position 45 has only a mild effect on reactivity, and that this is unlikely to be the site for electron transfer.     

Electron transport in hemoproteins. VIII. The effect of chemical modification of ferricytochrome C on the rate of its reduction by oxymyoglobin

The influence of chemical modification of separate amino acid residues in ferricytochrome c (Cyt c) on the rate of the redox reaction with MbO2 has been studied at various pH and ionic strength values. It is shown that alkylation of His-33 and Met-65 by bromacetate does not affect the reaction rate. On the contrary, acylation of Tyr-74 or one of the neighbouring lysines, Lys-72 or Lys-73, by the spin-label N-(2,2',5,5'-tetramethyl-3-carboxypyrrolin-1-oxy)-imidazol diminishes sharply the efficiency of electron transfer in the redox system studied. Besides, unlike the reaction between native proteins, the rate of electron transfer in this case does not depend on ionic strength. The modification of Tyr-74 or Lys 72/43 does not alter the midpoint potential and the entire conformation of Cyt c. The observed effects can therefore be explained by essential disturbance of interactions, first of all, the electrostatic ones in the active complex, which is induced by the attachment of the bulky reagent to the site of "active contact" of Cyt c. Based on the obtained findings and the atomic coordinates of Cyt c, the positions of all charge and some uncharged groups on the surface of Cyt c interacting with myoglobin during electron transfer are presented.     

Catalytic effect of ferricyanide between myoglobin and luminol and effect of temperature.

Specific catalytic oxidation of oxymyoglobin (MbO(2)) and luminol by ferricyanide was studied in a flow-injection system. MbO(2) in different redox states (ferric and ferrous) was oxidized to Mb(Fe(III)) by ferricyanide, and then specific binding of the ferrocyanide anion to Mb(Fe(III)) to the His 119 (GH1) region accelerated the electron transfer between Mb(Fe(III)) and luminol, which produced a chemiluminescence (CL) signal at 425 nm. The increased CL emission was correlated with the myoglobin concentration in the range 0.16-7.5 microg/mL. Thermogravimetry and differential scanning calorimetry were used to investigate the temperature effects on this reaction. The results showed that the CL intensity in the presence of myoglobin changed considerably with heating in the range 15-50 degrees C, and the maximal CL intensity was observed at 40 degrees C, corresponding to the glass transition temperature of myoglobin. The effect of different ligands and interferences were also studied.     

Electron transfer in hemoproteins. VIII. Influence of ionic strength on the rate of reduction of ferricytochrome c by oxymyoglobin derivatives, chemically modified at histidine residues

The influence of chemical modification of His residues in Mb on the rate of redox reaction in system MbO2--Cyt c has been studied at different ionic strengths and pH medium. The products of alkylation of all available His by bromacetate and iodacetamide, CM-Mb and CA-Mb, respectively, and myoglobin, modified by spin label 2,2', 6,6'-tetramethyl-4-bromoacetoxypiperidine-1-oxyl (SL) at His residue A10--Sl (His-A10)--Mb have been studied. It has been shown, that the character of the ionic strength dependence of reaction SL(His-A10)--MbO2 with Cyt c at pH 6.0 ann 7.0 is basically analogues to that, observed for intact protein. It means that only His-GH1 of two His residues, His-A10 and His-GH1, situated in the region of "active contact" of Mg with Cyt c molecule, participates in the interactions, essential for electron transfer. The interaction of the charge of this His with the negatively charged group of Cyt c is necessary, probably for the proper arrangement of other interactions in the active complex, because the deprotonation of His-GHl in the studied pH interval decreases the rate of the process by more than one order of magnitude. The rate of oxidation of MC-MbO2 and CA-MbO2 by ferricytochrome c, in contrast to intact protein, shows a weak dependence on the ionic strength and does not depend on the pH medium, throughout the range of ionic strengths from 0.005 to 1.0. The cause of the radical change in the ionic strength dependence is, probably, nearly entire disturbance of electrostatic interactions in the active complex due to chemical modification of His residues in the site of "active contact", and first of all, the His-CHl residue. The fact, that during alkylation of all available His in Mb the electron transfer persists in the system, points to that in the process of electron transfer to cytochrome c, uncharged group, most probably "inner" His-B5, participates. Based on the data on spatial structure and the obtained results, the positions of the charged groups in the site of "active contact" of Mb with Cyt c molecule are presented.     

Ferrocyanide carbon-13 NMR line broadening as a probe of electron transfer reactions

The electron transfer reaction between ferrocyanide ion and the blue copper protein, stellacyanin, has been investigated by means of 13C NMR line broadening of the inorganic oxidant. The temperature dependence of the ferrocyanide line broadening gives an activation energy for the electron transfer reaction of 17 +/- 3 kJ. The apparent rate constant decreases with increasing concentration of K4Fe(CN)6, a result which can be explained either by formation of a strong precursor ferrocyanide--stellacyanin [Cu(II)] complex or by increased formation of KFe(CN)3-6 ion pairs. The direct electron transfer between ferrocyanide and ferricyanide has also been studied by 13C NMR line broadening of the former species. The ferricyanide concentration dependence of the exchange line broadening yields a value for the apparent second-order rate constant at 25 degrees C of k = 1.65 . 10(3) M-1 . s-1, in agreement with previously reported values derived from 14N NMR and isotope exchange studies. This rate constant shows a linear dependence on the K+ concentration, independent of ionic strength, a result which confirms the importance of ion pair species such as KFe(CN)3-6 and KFe(CN)2-6 in the direct electron transfer mechanism. The general applications of the method are discussed, including the considerations which suggest that a wide range of electron transfer rates, from about 1 s-1 to 4 . 10(3) s-1, are, in principle, accessible to this technique. The potential utility of ferrocyanide 13C spin--lattice relaxation time measurements is decreasing the lower limit of this range is also discussed.     

Transient kinetics of electron transfer reactions of flavodoxin: ionic strength dependence of semiquinone oxidation by cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid and computer modeling of reaction complexes

Electron transfer reactions between Clostridum pasteurianum flavodoxin semiquinone and various oxidants [horse heart cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic [horse heart cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid (EDTA)] have been studied as a function of ionic strength by using stopped-flow spectrophotometry. The cytochrome c reaction is complicated by the existence of two cytochrome species which react at different rates and whose relative concentrations are ionic strength dependent. Only the faster of these two reactions is considered here. At low ionic strength, complex formation between cytochrome c and flavodoxin is indicated by a leveling off of the pseudo-first-order rate constant at high cytochrome c concentration. This is not observed for either ferricyanide or ferric EDTA. For cytochrome c, the rate and association constants for complex formation were found to increase with decreasing ionic strength, consistent with negative charges on flavodoxin interacting with the positively charged cytochrome electron transfer site. Both ferricyanide and ferric EDTA are negatively charged oxidants, and the rate data respond to ionic strength changes as would be predicted for reactants of the same charge sign. These results demonstrate that electrostatic interactions involving negatively charged groups are important in orienting flavodoxin with respect to oxidants during electron transfer. We have also carried out computer modeling studies of putative complexes of flavodoxin with cytochrome c and ferricyanide, which relate their structural properties to both the observed kinetic behavior and some more general features of physiological electron transfer processes. The results of this study are consistent with the ionic strength behavior described above.     

Partitioning of electrostatic and conformational contributions in the redox reactions of modified cytochromes c.

The reduction of acetylated, fully succinylated and dicarboxymethyl horse cytochromes c by the radicals CH3CH(OH), CO2.-, O2.-, and e-aq' and the oxidation of the reduced cytochrome c derivatives by Fe(CN)3-6 were studied using the pulse radiolysis technique. Many of the reactions were also examined as a function of ionic strength. By obtaining rate constants for the reactions of differently charged small molecules redox agents with the differently charged cytochrome c derivatives at both zero ionic strength and infinite ionic strength, electrostatic and conformational contributions to the electron transfer mechanism were effectively partioned from each other in some cases. In regard to cytochrome c electron transfer mechanism, the results, especially those for which conformational influences predominate, are supportive of the electron being transferred in the heme edge region.     

Mechanism of electron transfer between myoglobin derivatives and ferricytochrome C

Progress in the studies of the electron transport mechanism in biological systems is greatly hindered by the lack of detailed structural information about the components of these systems. That is why a study of electron transfer between protein molecules with the known spatial organization in model reactions in vitro is of great importance. In this respect the MbO2--Cyt C oxidation-reduction reaction offers unique possibilities. Studies of the effects of pH and ionic strength of the medium on the kinetics of this reaction in combination with chemical modification of single amino acid residues of Mb and Cyt C enabled us to identify those parts of the surface of haemoproteins where the molecules come into "active contact". A variation in the number or/and the arrangement of the charged groups at the "active sites" of the molecules induced by both changing the medium pH and chemical modification of some of these groups lowers markedly the probability of electron transfer in the system (e.g. His GH1 and His A10 in Mb) or blocks it entirely (acylation of Lys 72 (73) or Tyr 74 in Cyt C). Based on the results obtained and on the data of Mb and Cyt C X-ray analysis, the figures of spatial arrangement of the groups at the "active sites" of these molecules are presented.     

Photoinduced electron transfer between cytochrome c peroxidase and horse cytochrome c labeled at specific lysines with (dicarboxybipyridine)(bisbipyridine)ruthenium(II)

The reactions of yeast cytochrome c peroxidase with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)(bisbipyridine)ruthenium(II) [Ru(II)] were studied by flash photolysis. All of the derivatives formed complexes with cytochrome c peroxidase compound I (CMPI) at low ionic strength (2 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a short laser flash resulted in electron transfer to the ferric heme group in cytochrome c, followed by electron transfer to the radical site in CMPI. This reaction was biphasic and the rate constants were independent of CMPI concentration, indicating that both phases represented intracomplex electron transfer from the cytochrome c heme to the radical site in CMPI. The rate constants of the fast phase were 5200, 19,000, 55,000, and 14,300 s-1 for the derivatives modified at lysines 13, 25, 27, and 72, respectively. The rate constants of the slow phase were 260, 520, 200, and 350 s-1 for the same derivatives. These results suggest that there are two binding orientations for cytochrome c on CMPI. The binding orientation responsible for the fast phase involves a geometry that supports rapid electron transfer, while that for the slow phase allows only slow electron transfer. Increasing the ionic strength up to 40 mM increased the rate constant of the slow phase and decreased that of the fast phase. A single intracomplex electron transfer phase with a rate constant of 2800 s-1 was observed for the lysine 72 derivative at this ionic strength. When a series of light flashes was used to titrate CMPI to CMPII, the reaction between the cytochrome c derivative and the Fe(IV) site in CMPII was observed. The rate constants for this reaction were 110, 250, 350, and 140 s-1 for the above derivatives measured in low ionic strength buffer.     

Ionic strength and pH dependence of the reaction between plastocyanin and Photosystem 1. Evidence of a rate-limiting conformational change

The electron-transfer reaction between spinach wild-type plastocyanin (Pc(WT)) two site-directed mutants, Pc(Thr79His) and Pc(Lys81His), and spinach Photosystem 1 particles, has been studied as a function of protein concentration, ionic strength and pH by using laser-flash absorption spectroscopy. The kinetic data are interpreted using the simplest possible three-step model, involving a rate-limiting conformational change preceding intracomplex electron transfer. The three proteins show similar concentration, pH and ionic strength dependencies. The effects of ionic strength and pH on the reaction indicate a strong influence of complementary charges on complex formation and stabilization. Studies with apoprotein support the opinion that the hydrophobic patch is critical for an productive interaction with the reaction center of Photosystem 1. Together with earlier site-directed mutagenesis studies, the absence of a detectable Photosystem 1 reaction in the presence of reduced azurin, stellacyanin, cytochrome c and cytochrome c₅₅₁, demonstrates the existence of a high level of specificity in the protein-protein interface in the formation of an efficient electron-transfer complex.     

Site-directed mutagenesis of yeast cytochrome c peroxidase shows histidine 181 is not required for oxidation of ferrocytochrome c

The long-distance electron transfer observed in the complex formed between ferrocytochrome c and compound I, the peroxide-oxidized form of cytochrome c peroxidase (CCP), has been proposed to occur through the participation of His 181 of CCP and Phe 87 of yeast iso-1 cytochrome c [Poulos, T. L., & Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330]. We have examined the role of His 181 of CCP in this process through characterization of a mutant CCP in which His 181 has been replaced by glycine through site-directed mutagenesis. Data from single-crystal X-ray diffraction studies, as well as the visible spectra of the mutant CCP and its 2-equiv oxidation product, compound I, show that at pH 6.0 the protein is not dramatically altered by the His 181----Gly mutation. The rate of peroxide-dependent oxidation of ferrocytochrome c by the mutant CCP is reduced only 2-fold relative to that of the parental CCP, under steady-state conditions. Transient kinetic measurements of the intracomplex electron transfer rate from ferrous cytochrome c to compound I indicate that the rate of electron transfer within the transiently formed complex at high ionic strength (mu = 114 mM, pH = 6) is also reduced by approximately 2-fold in the mutant CCP protein. The relatively minor effect of the loss of the imidazole side chain at position 181 on the kinetics of electron transfer in the CCP-cytochrome c complex precludes an obligatory participation of His 181 in electron transfer from ferrous cytochrome c to compound I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hemoglobin and myoglobin as reducing agents in biological systems. Redox reactions of globins with copper and iron salts and complexes

In addition to reversible O₂ binding, respiratory proteins of the globin family, hemoglobin (Hb) and myoglobin (Mb), participate in redox reactions with various metal complexes, including biologically significant ones, such as those of copper and iron. HbO₂ and MbO₂ are present in cells in large amounts and, as redox agents, can contribute to maintaining cell redox state and resisting oxidative stress. Divalent copper complexes with high redox potentials (E ₀, 200-600 mV) and high stability constants, such as [Cu(phen)₂]²⁺, [Cu(dmphen)₂]²⁺, and CuDTA oxidize ferrous heme proteins by the simple outer-sphere electron transfer mechanism through overlapping π-orbitals of the heme and the copper complex. Weaker oxidants, such as Cu²⁺, CuEDTA, CuNTA, CuCit, CuATP, and CuHis (E ₀≤ 100-150 mV) react with HbO₂ and MbO₂ through preliminary binding to the protein with substitution of the metal ligands with protein groups and subsequent intramolecular electron transfer in the complex (the site-specific outer-sphere electron transfer mechanism). Oxidation of HbO₂ and MbO₂ by potassium ferricyanide and Fe(3) complexes with NTA, EDTA, CDTA, ATP, 2,3-DPG, citrate, and pyrophosphate PP_i proceeds mainly through the simple outer-sphere electron transfer mechanism via the exposed heme edge. According to Marcus theory, the rate of this reaction correlates with the difference in redox potentials of the reagents and their self-exchange rates. For charged reagents, the reaction may be preceded by their nonspecific binding to the protein due to electrostatic interactions. The reactions of LbO₂ with carboxylate Fe complexes, unlike its reactions with ferricyanide, occur via the site-specific outer-sphere electron transfer mechanism, even though the same reagents oxidize structurally similar MbO₂ and cytochrome b ₅ via the simple outer-sphere electron transfer mechanism. Of particular biological interest is HbO₂ and MbO₂ transformation into met-forms in the presence of small amounts of metal ions or complexes (catalysis), which, until recently, had been demonstrated only for copper compounds with intermediate redox potentials. The main contribution to the reaction rate comes from copper binding to the “inner” histidines, His97 (0.66 nm from the heme) that forms a hydrogen bond with the heme propionate COO^– group, and the distal His64. The affinity of both histidines for copper is much lower than that of the surface histidines residues, and they are inaccessible for modification with chemical reagents. However, it was found recently that the high-potential Fe(3) complex, potassium ferricyanide (400 mV), at a 5 to 20% of molar protein concentration can be an efficient catalyst of MbO₂ oxidation into metMb. The catalytic process includes binding of ferrocyanide anion in the region of the His119 residue due to the presence there of a large positive local electrostatic potential and existence of a “pocket” formed by Lys16, Ala19, Asp20, and Arg118 that is sufficient to accommodate [Fe(CN)₆]^4–. Fast, proton-assisted reoxidation of the bound ferrocyanide by oxygen (which is required for completion of the catalytic cycle), unlike slow [Fe(CN)₆]^4– oxidation in solution, is provided by the optimal location of neighboring protonated His113 and His116, as it occurs in the enzyme active site.     

The oxidation of ferrocytochrome c in nonbinding buffer.

The apparent equilibrium constant and rate of oxidation was investigated for the reaction of cytochrome c with iron hexacyanide. It was found that if horse heart ferricytochrome c was exposed to ferricyanide (to oxidize traces of reduced protein) the cytochrome subsequently, even after extensive dialysis, had an apparent equilibrium constant different from that of electrodialyzed protein. The effect of ferricyanide ion apparently cannot be removed by ordinary dialysis. The ionic strength dependence of the apparent equilibrium constant and bimolecular oxidation rate constant was measured in the range 1--200 mM using Tris--cacodylate or potassium phosphate buffers at pH 7.0, and electrodialyzed horse heart cytochrome c. The oxidation reaction proceeded very rapidly. Extrapolated to zero ionic strength, kox (approximately 9 X 10(9) M-1 S-1) was about 7% of that calculated for a diffusion-limited reaction. Since the exposed heme edge occupies only the order of 3% of the surface area, electron transfer apparently results at nearly every collision with the active-site region. An effective charge of + 7.8 units was estimated for the oxidation reaction. The rate of oxidation of Pseudomonas aeruginosa c551 was much slower (kox at mu = 0 was the order of 6 X 10(3)), and was not consistent with diffusion-limited kinetics.