Ferrocyanidea novel catalyst for oxymyoglobin ... - Wiley Online Library

Ferrocyanide ) a novel catalyst for oxymyoglobin oxidation by molecular oxygen G. B. Postnikova, S. A. Moiseeva, E. A. Shekhovtsova, E. V. Goraev and V. S. Sivozhelezov Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Russia

Keywords catalysis, ferrocyanide, modification, myoglobin, oxidation Correspondence G. B. Postnikova or V. S. Sivozhelezov, Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia Fax: +7 4967 330509 Tel: +7 4967 739155 E-mail: [email protected], [email protected]; [email protected] (Received 13 March 2007, revised 4 June 2007, accepted 22 August 2007) doi:10.1111/j.1742-4658.2007.06061.x

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 fi 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.

It is well known that oxidation of the respiratory proteins, hemoglobin and myoglobin, by oxygen is markedly enhanced by small amounts of divalent copper compounds with moderate redox potentials (E0 of approximately 100–150 mV, copper catalysis) [1–4]. The catalytic Reaction follows the site-specific outersphere electron transfer mechanism, which includes the preliminary binding of the copper reagent to histidine residues. Until recently, the compounds of other metals with middle and moreover high redox potentials were not considered to be the effective catalysts because they are able to oxidize hemoglobin and myoglobin through direct interaction with the heme group (the simple outer-sphere electron transfer mechanism) and their reduced forms, as distinct from those of copper, are very slowly reoxidized by dissolved oxygen [1–5].

We have found, however, that the small amounts (5–20% of concentration of the protein) of potassium ferricyanide, which is the complex of iron and a strong oxidant, can catalyze oxidation of sperm whale oxymyoglobin [6,7]. The reaction rate is markedly accelerated upon acidification of the solution in the pH 5–8 range. Potassium ferricyanide (E0 of the [Fe(CN)6]3– ⁄ [Fe(CN)6]4– couple is 415–430 mV) is widely used for fast oxidation of heme proteins. Reduced ferrocyanide formed in the reaction, as well as other salts of divalent iron, is reoxidized very slowly, even in acids [8]. The cause of the catalysis is demonstrated to be specific complexing of ferrocyanide anion with myoglobin [6,7]. The [Fe(CN)6]4– ion binds at the protein surface in the region of His119, approximately 0.2 nm from the heme plane, with a subsequent electron transfer

Abbreviations CM-Mb, carbyxymethylated metmyoglobin; CM-MbO2, carbyxymethylated oxymyoglobin; ESP, electrostatic potential; [Fe(CN)6]4–, ferrocyanide; MbO2, oxymyoglobin; met-Mb, metmyoglobin.

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FEBS Journal 274 (2007) 5360–5369 ª 2007 The Authors Journal compilation ª 2007 FEBS

G. B. Postnikova et al.

from the heme iron (the site-specific outer-sphere mechanism of electron transfer). Thus, at low concentrations of ferricyanide, two different processes simultaneously occur: (a) rapid oxidation of the correspondent quantity of oxymyoglobin by interaction [Fe(CN)6]3– with the heme according to simple outersphere electron transfer mechanism and (b) slower transformation of the remaining protein in metmyoglobin (met-Mb) along the catalytic path. If ferrocyanide is added to oxymyoglobin, only the catalysis is observed. In sperm whale myoglobin, the His12, His113 and His116 residues, along with His119, are located at the site where the complex with [Fe(CN)6]4– is formed. The protonation of these histidines in the pH 5–8 range must facilitate the binding of [Fe(CN)6]4– due to creation of a higher local positive electrostatic potential (ESP) there. Only ferrocyanide bound to myoglobin, unlike the same anion in solution, is capable of being quickly reoxidized by dissolved oxygen (with participation of protons), which is necessary for closing the catalytic cycle. In the present study, the molecular mechanism of the ferrocyanide-catalyzed oxidation of oxymyoglobin (MbO2) is studied in detail by a comparison of the kinetics of the redox reaction for native oxymyoglobins from sperm whale, horse and pig, the chemically modified sperm whale MbO2, with all accessible His residues alkylated by sodium bromoacetate (CMMbO2), and the mutant protein with His119 replaced by aspartic acid [MbO2(His119 fi Asp)]. Sperm whale, horse and pig myoglobins have highly homologous sequences and 3D structures, and identical redox potentials, but differ markedly in the quantity of histidines on their surface and the overall charge of the molecule. The effect of pH, ionic strength, concentration of the catalyst and complexing His119 to redoxinactive zinc ion on the reaction rate is investigated. The distribution of ESP around the native and mutant myoglobin molecules are calculated at different pH values in the pH 5–8 range. The ESP distributions in the [Fe(CN)6]4–-binding site of various samples are analysed. The steric features of myoglobin surface are investigated relative to the presence of hollows and cavities capable of accommodating an anion of ferrocyanide-like size.

Results Characteristics of myoglobins studied In the pH 5–8 range investigated, sperm whale Mb (pI 8.3) is positively charged, whereas the overall charge of horse Mb (pI 7.4) and pig Mb (pI 7.2)

Ferrocyanide catalysis of oxymyoglobin oxidation

changes from positive to negative values. In horse myoglobin, in contrast to the sperm whale protein, the solvent-exposed His12 is replaced by Gln and, in pig myoglobin, three residues, His12, His113 and His116, are also replaced by Gln [9,10]. Another 19 replacements in these native proteins are identical or homologous except for the replacement of positively charged Lys87(F2) of sperm whale and horse Mb by neutral Thr87 in pig Mb. This residue is located far from the place where [Fe(CN)6]4– is bound. Note that the amino acid composition of the heme cavity is strictly invariant in all three proteins. Isoelectric focusing of carbyxymethylated metmyoglobin (CM-Mb) in polyacrylamide gel yields a single wide band with pI 5.2, so that carboxymethylated sperm whale Mb is neutral or negative in the pH 5–8 range. The negative charge of CM-Mb is due to addition of negative CH2COO– groups to His residues. All solvent-accessible histidines can react with bromoacetate, His12, 13, 116 and 81, forming predominantly disubstituted derivatives that cannot be protonated in the pH 5–8 range, whereas His48 and 119 monoalkylated compounds are capable of accepting protons [11,12]. The absorption and CD spectra in the UV and visible regions, as well as the thermal stability of CM-Mb and intact Mb, do not differ. The single replacement of surface His119 with Asp in the mutant Mb has no effect on the conformation of the heme cavity and protein molecule as a whole because the differences in the UV and visible absorption spectra, the pK values of the met-hydroxy transition, and the stability of the protein conformation in the pH 6.0–12.5 range, are within the limits of experimental error (Table 1). According to the isoelectric focusing data, the isoelectric point of native met-Mb (pI 8.3) and mutant met-Mb (pI 8.2) also changes insignificantly, which reflects the change in the total charge of met-Mb within approximately 0.5 charge units caused by mutation. Kinetics of oxymyoglobin oxidation at the presence of ferrocyanide Figure 1 shows the dependence of oxidation rates of the native sperm whale, horse and pig MbO2, as well as modified sperm whale CM-MbO2 and MbO2(His119 fi Asp), on the catalyst concentration under conditions when it is much lower than the protein concentration (up to 20% of the latter). A linear dependence of the oxidation rate on [Fe(CN)6]4– concentration is observed for all proteins studied (curves 1–5), with the lowest rate being for pig MbO2 and CM-MbO2 (curves 3 and 4). At higher catalyst


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Table 1. Spectral properties, pK values of met-hydroxy transition, and pH of alkaline denaturation of sperm whale met-Mb and mutant metMb(His119 fi Asp).

Sample

kSoret (nm)

ISoret ⁄ I505

ISoret ⁄ I280

I280 ⁄ I505

pK of met-hydroxy transition

pH of alkaline denaturation

Native met-Mb Mb[His119(r)Asp]

409.5 409.6

16.5 16.6

5.03 4.96

3.3 3.4

8.9 8.9

11.5 11.4

Fig. 1. Dependence of the oxidation rate of native sperm whale MbO2 (1), horse MbO2 (2), pig MbO2 (3), chemically modified CM-MbO2 (4) and mutant MbO2(His119 fi Asp) from sperm whale (5) on the ferrocyanide concentration under conditions where it is much lower compared to that of the protein (0.01 M Tris-malate buffer, pH 5.2, 20 C. Protein concentration is 22.5 lM).

Fig. 2. Dependence of the oxidation rate of native sperm whale MbO2 (1), horse MbO2 (2) and pig MbO2 (3), as well as of modified CM-MbO2 (4) and mutant MbO2(His119 fi Asp) (5), on the [Fe(CN)6]4– concentration under conditions of its excess relative to the protein concentration (0.01 M Tris-malate buffer, pH 6.4, 20 C. Protein concentration is 22.5 lM).

concentrations (Fig. 2), for the [Fe(CN)6]4– ⁄ [MbO2] ratios from 1 to 50, the rates of oxidation of the native sperm whale and horse MbO2 are approximately 5362

Fig. 3. The pH-dependence of oxidation rate of sperm whale MbO2 (1), horse MbO2 (2), pig MbO2 (3), CM-MbO2 (4) and MbO2(His119 fi Asp) (5) in the presence of potassium ferrocyanide (5% of the protein concentration) (0.01 M Tris-malate buffer, 20 C. Protein concentration is 22.5 lM).

identical and reach saturation at a ratio greater than 30 : 1 (curves 1 and 2). For pig and CM-MbO2, the oxidation rates are also similar (Fig. 2, curves 3 and 4), but they increase much less in relation to [Fe(CN)6]4– concentration than for sperm whale and horse MbO2, and neither reaches saturation. At the [Fe(CN)6]4– ⁄ [MbO2] ratio of 50 : 1, they are approximately five-fold lower compared to those of native sperm whale and horse proteins. The rate of mutant MbO2(His119 fi Asp) oxidation (Fig. 2, curve 5) also increases more slowly with the catalyst concentration compared to that of native sperm whale and horse MbO2 (two- or three-fold), but much faster than that of pig and CM-MbO2. Figure 3 shows the effect of pH within the pH 5–8 range on the rate of oxidation of all MbO2 studied in the presence of 5% potassium ferrocyanide (relative to the protein concentration). The oxidation rates of native sperm whale and horse MbO2 differ insignificantly, and the shape of the pH-dependence curve is identical in both cases: the oxidation rate increases at pH < 7 (Fig. 3, curves 1 and 2). By contrast, the rate of pig MbO2 oxidation does not depend on pH at all and is very low in the pH range studied (Fig. 3, curve 3). The rate of CM-MbO2 oxidation also remains low in the




Fig. 4. Effect of ionic strength on the rate of oxidation of sperm whale MbO2 (1), horse MbO2 (2), CM-MbO2 (3) and MbO2(His119 fi Asp) (4) in the presence of potassium ferrocyanide (5% of the protein concentration) (0.01 M Tris-malate buffer, KCl, pH 5.1, 20 C. Protein concentration is 22.5 lM).

pH 5–8 range, but increases slightly at pH < 5.5 (Fig. 3, curve 4). In the case of mutant myoglobin, the increase in the reaction rate at pH < 5.5 is much more significant (Fig. 3, curve 5) than for pig and CM-MbO2. The oxidation of MbO2 at a pH < 5.5 is typically indicative for its autooxidation [13]. The [Fe(CN)6]4–-catalyzed oxidation of intact sperm whale and horse MbO2 is strongly inhibited as the ionic strength increases from 0 to 0.55 (pH 5.1), especially at low (I ¼ 0–0.1) ionic strengths (Fig. 4, curves 1 and 2), which indicates an important role for electrostatic interactions in the reaction mechanism. At higher ionic strengths (I > 0.1), the reaction rate is low throughout the pH 5–8 range. The rate of CMMbO2 oxidation is low and independent of ionic strength (Fig. 4, curve 3). An insignificant increase at high I can be attributed to the negative charge of both the reagent and protein, so that charge screening can facilitate their interaction. The rate of pig MbO2 oxidation is also very low and does not depend on ionic strength (data not shown). The oxidation rate of

MbO2[His119 fi Asp] is decreased as the ionic strength increased at low I (Fig. 4, curve 4), although the dependence is not as pronounced as it is for native sperm whale and horse proteins. The addition of zinc ions into reaction medium at the [Zn2+] : [MbO2] ratio of 2, when only one site at the His119 region is saturated, strongly inhibits the oxidation of native sperm whale and horse MbO2, even in large excess of the catalyst (Table 2). No substantial effect of zinc is observed on the reaction rate of pig MbO2, as well as carboxymethylated and mutant sperm whale MbO2, for an up to 50-fold excess of the catalyst. The high affinity of Zn2+ to the His119(GH1) site of myoglobin is explained by formation of the chelate complex, in which functional groups of nearby Lys16(A14) and Asp122(GH4) participate [14]. As demonstrated previously, the binding of redox-inactive zinc ion to sperm whale MbO2 (E0 of the Zn2+ ⁄ Zn couple is 763 mV) does not influence the total protein conformation and the rate of its autooxidation relative to native MbO2 [15]. Oxidation of ferrocyanide in the presence of metmyoglobin Addition of ferrocynide to metmyoglobin in the presence of oxygen leads to its oxidation to ferricyanide. Indeed, after metMb was incubated for 30 min with ferrocyanide under catalysis-optimal conditions (0.01 m buffer, pH 6.2) and then chromatographed on Sephadex CM-25 column equilibrated with the same buffer, the protein becomes absorbed whereas ferricyanide is released, identified by its yellow color and the typical wide spectrum in the visible region. ESP distribution around the myoglobin molecule The strong dependence of the ferrocyanide-catalyzed oxidation rate on ionic strength (Fig. 4) suggests the important role of electrostatic interactions in the reac-

Table 2. Effect of zinc ions on the rate of oxidation of native sperm whale MbO2, carboxymethylated CM-MbO2 and mutant MbO2(His119 fi Asp) (m0 · 106, MÆmin)1) at different catalyst ⁄ protein ratios (0.01 M Tris-malate buffer, pH 6.4, 20 C). [[Fe(CN)6]4–] ⁄ [MbO2] ¼ 5 : 1 [Zn2+]: [MbO2]

[[Fe(CN)6]4–] ⁄ [MbO2] ¼ 10 : 1 [Zn2+]: [MbO2]

[[Fe(CN)6]4–] ⁄ [MbO2] ¼ 50 : 1 [Zn2+]: [MbO2]

Sample

–

2:1

Inhibition factor

–

2:1

Inhibition factor

–

2:1

Inhibition factor

Sperm whale Mb Horse Mb Pig Mb CM-Mb Mb[His119(r)Asp]

15.0 34.8 1.5–2.0 2.03 7.20

2.5 3.3–3.9 1.0 2.3 4.6

6 10 1.5–2 1 1.55

27.5 34.8 1.5–2.0 2.55–3.36 10.7

2.8 3.3–3.9 1.0 3.23–3.3 6.2

10 10 1.5–2

58.2 64.0 12.1 – –

2.3 2.6 1.5 – –

25 25 8 –


1.5

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A


B

C

Fig. 5. ESP distribution around the myoglobin molecule at the experimentally found site of [Fe(CN)6]4– linkage in the His119 region for two pH values, 5.2 (top) and 7.5 (bottom), (A) In sperm whale myoglobin; (B) in pig myoglobin and (C) in mutant Mb(His119 fi Asp). Red, area of negative potential (< )0.3 kTÆe)1); blue, area of positive potential (+0.3 > kTÆe)1); white, area of a potential close to neutral (from )0.3 to +0.3 kTÆe)1).

tion mechanism. The inner ligand sphere of iron in [Fe(CN)6]4– is completely filled with strong CN– ligands that cannot be displaced by the protein groups, so that ferrocyanide can bind to the protein only at the expense of electrostatic and, probably, steric interactions. In this connection, the distributions of ESP around myoglobin molecules studied are calculated in the pH 5–8 range. Figure 5A shows the ESP distribution in the experimentally determined [Fe(CN)6]4– binding site of native sperm whale Mb, calculated at pH 5.2 and 7.5. For horse Mb, the distributions are practically identical, differing by only a small amount at pH 5.2 in the region, where His12 is replaced by Gln. At pH < 6, the catalyst-binding site in both myoglobins is in a field of large local positive potential. This potential becomes neutral at pH > 7 because of deionization of His10, His113, His116 and His119 located there (the ionization state of other charged groups does not vary). The catalyst-binding site of pig Mb at pH 5.2 (Fig. 5B) is also in the region of positive ESP, although slightly smaller than in sperm whale and horse myoglobins, as His113 and His116 are replaced by Gln. Note that replacement of His119 by Asp in the mutant myoglobin changes a positive potential in the site of [Fe(CN)6]4– binding, typical for acid pH, to a negative one (Fig. 5C), which should make complex5364

ing anionic catalysts unfavorable. The positive ESP is preserved only at the His113 and His116 regions. The local ESP of this site becomes fully negative at pH>7. Hollows and cavities in the myoglobin molecule The inhibiting effect of Zn2+ on ferrocyanide-catalysed oxidation of sperm whale and horse myoglobins highlights the relevant value not only of electrostatic, but also steric interactions in the reaction mechanism, because positive ESP at the catalyst-binding site with Zn2+ is fully maintained (or even increased). It is obviously of great importance also for the optimal dynamics of the protein groups located at the [Fe(CN)6]4– binding site, which is substantially changed upon chelating Zn2+. In the crystal structure of sperm whale Mb, ten hollows inside the molecule and fourteen cavities at its surface are found [16,17]. With the exception of the heme pocket, only three cavities have linear dimensions of 20–24 A˚ and a volume of 12–94 A˚3, which are sufficient for at least partial accommodation of ferrocyanide (B ¼ 9 A˚, V 165 A˚3). None of them is in the region of positive ESP, including histidines that are experimentally shown to be important for the catalysis. In the structure of Mb in solution, one more cavity near His119 is found (Fig. 6, top). It is formed by




Lys16, Ala19, Asp20, His24 and Arg118 and can easily accommodate ferrocyanide, if His119 is in an ‘open conformation’ (Fig. 6, bottom). For the ferri-ferrocyanide ion that is strongly charged, relatively bulky, and not a natural ligand of myoglobin, a pre-existing cavity is essential for binding to avoid distortion of the 3D structure of myoglobin. The amino acids forming the cavity are invariant for 66 different animal myoglobins (in pig myoglobin, Arg118 is replaced by homologous Lys118). This cavity is closed in the crystal Mb structure, as His119, which is H-bonded to inner His24, faces into the interior of the molecule. It is also closed is in the Zn2+–Mb chelate complex involving functional groups of His119, Lys16 and Asp122. By contrast, in mutant MbO2(His119 fi Asp), the cavity is permanently open, which allows [Fe(CN)6]4– ions to freely enter, although the energy of their binding must be approximately 4 kcal less than in the native protein [17].

Discussion The scheme of ferrocyanide-catalyzed oxidation of oxymyoglobin involves specific binding of ferrocyanide to the protein (Reaction 1), rapid oxidation of bound [Fe(CN)6]4– by dissolved O2 mediated by protons (Reaction 2) and formation of the resulting products (Reaction 3) [6,7]: k1 4 Mb(II)O2 þ ½FeII(CN)6 4 ! Mb(II)O2 ½FeII(CN)6 k1 ð1Þ k2 Mb(II)O2 [FeII(CN)6 4 þ O2 þ Hþ ! Mb(II)O2 [FeIII(CN)6 3 þ HO2

ð2Þ

k3 Mb(II)O2 [FeIII(CN)6 3 þ H2 O ! Mb(III)H2 O þ [FeII(CN)6 4 þ O2

ð3Þ

Rate constant k3 is an effective one comprising several individual kinetic and equilibrium constants, with all these reactions being rapid and not limiting the process. An expression for the initial reaction rate (t0) is received by solving the set of Eqns 1–3 according to the formal kinetic schemes for homogeneous catalysis in solution under stationary conditions (Eqn 4), and well describes the linear dependence of t0 on the [Fe(CN)6]4– and proton concentration in excess of MbO2 relative to ferrocyanide [6,7]: t0 ¼ k2 ½MbO2 0 ½½Fe(CN)6 4 0 ½O2 ½Hþ =Kdis where Kdis ¼ k)1 ⁄ k1.

ð4Þ

In excess of ferrocyanide, [[Fe(CN)6]4–] >> [MbO2], the dependence of t0 on the [Fe(CN)6]4– concentration (with a saturation) is similar to those for enzymatic kinetics, where kcat ¼ k2 [O2][H+], Vmax ¼ kcat [MbO2]0, and Kdis(obs) ¼ (k2 [O2][H+] + k)1) ⁄ k2: t0 ¼ k2 ½MbO2 0 ½½Fe(CN)6 4 0 ½O2 ½Hþ = ðk1 þ k2 ½O2 ½Hþ Þ=ðk1 þ ½½Fe(CN)6 4 0 Þ

ð5Þ

If, as usual, k)1 >> k2 [O2][H+], a simpler expression is obtained: t0 ¼ k2 ½MbO2 0 ½½Fe(CN)6 4 0 ½O2 ½Hþ = ðKdis þ ½½Fe(CN)6 4 0 Þ

ð6Þ

Table 3 presents the values of the equilibrium and kinetic constants, Kdis ¼ k)1 ⁄ k1 and k2, determined from the experimental data according to the Eqns 4 and 6, in the pH 5.0–8.0 range [6,7]. The strong pH dependence of Kdis (Table 3) and the dependence of the catalytic rate on ionic strength (Fig. 4) indicate that the [Fe(CN)6]4– binding at the His119 region is largely mediated by electrostatic interactions. It is also supported by the large local positive ESP (at pH < 6) at the catalyst-binding site (Fig. 5A`). Apparently, the protonation not only of His119, but also His113 and 116 located nearby is important for formation of an effective catalytic complex. The His12 charge is not necessary for the binding because its absence in horse Mb does not influence Kdis. The fact that local ESP varies from highly positive to neutral–negative due to deprotonation of His113, 116 and 119 in the pH 5–8 range explains well both the sharp reduction of Kdis and the oxidation rates of sperm whale and horse MbO2 (Fig. 3, curves 1 and 2). Note that alterations in the [Fe(CN)6]4– affinity to Mb is irrelevant to pH-dependent changes of the overall protein charge because Kdis values are identical at the same pH for sperm whale Mb (pI 8.3) and horse Mb (pI 7.4). The selectivity of [Fe(CN)6]4– complexing at the His119 region is mediated, on the other hand, by a local cavity capable of accommodating ferrocyanide anion. The cavity is inaccessible for ferrocyanide in the Zn2+–Mb chelate involving functional groups of His119, Lys16 and Asp122, thus well explaining the strong inhibiting effect of Zn2+ ions. The mean Kdis for Zn2+ in the pH 5.0–8.0 range is 2.3 · 106 m [5]; thus, its binding here is 100-fold stronger compared to [Fe(CN)6]4–, particularly at pH > 6, so even very high concentrations of ferrocyanide cannot compete with zinc. The crucial role of ferrocyanide binding to myoglobin is confirmed by a considerable inhibition of the


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Table 3. Equilibrium and kinetic parameters of ferrocyanide-catalyzed oxidation of native sperm whale MbO2, carboxymethylated CM-MbO2, and mutant MbO2[His119 fi Asp]. Experimental conditions

Myoglobin

pH

[H+],

M

[Fe(CN)6]4–] >> [MbO2]

Sperm whale

[Fe(CN)6]4–]