Aplysia oxymyoglobin with an unusual stability property

Native oxymyoglobin was isolated directly from the radular muscle of Aplysia kurodai with complete separation from metmyoglobin on a DEAE-cellulose column. It was examined for its spectral and stability properties. The spectrum of Aplysia MbO2 , which lacks the distal histidine, is very similar to those of mammalian oxymyoglobins , the alpha-peak being higher than the beta-peak and the absorbance ratio being 1.03. Its stability, however, is quite different from those of the mammalian oxymyoglobins , and Aplysia MbO2 is found to be extremely susceptible to autoxidation. Its rate is one-hundred times higher at pH 9.0, and its pH dependence is unusual and much less steep, when compared with sperm whale MbO2 as reference.     

Amino acid sequence of myoglobin from the mollusc Dolabella auricularia

The complete amino acid sequence of the myoglobin from Dolabella auricularia, a common gastropodic mollusc on the Japanese coast, has been determined. The myoglobin is composed of 146 amino acid residues, is acetylated at the NH2 terminus, and contains a single histidine residue at position 95 which most likely corresponds to the heme-binding proximal histidine. The sequence of Dolabella myoglobin shows strong homology (72-77%) with those of Aplysia myoglobins. The autoxidation rate of Dolabella oxymyoglobin (MbO2) was examined in 0.1 M buffer at 25 degrees C over pH range 4.8-12. Dolabella MbO2 was extremely unstable between pH 7 and 11, and the pH dependence of the stability was quite different from that of sperm whale MbO2. This property may be partly due to the absence of a distal (E7) histidine in Dolabella myoglobin.     

Stability of yeast iso-1-ferricytochrome c as a function of pH and temperature.

Absorbance-detected thermal denaturation studies of the C102T variant of Saccharomyces cerevisiae iso-1-ferricytochrome c were performed between pH 3 and 5. Thermal denaturation in this pH range is reversible, shows no concentration dependence, and is consistent with a 2-state model. Values for free energy (delta GD), enthalpy (delta HD), and entropy (delta SD) of denaturation were determined as functions of pH and temperature. The value of delta GD at 300 K, pH 4.6, is 5.1 +/- 0.3 kcal mol-1. The change in molar heat capacity upon denaturation (delta Cp), determined by the temperature dependence of delta HD as a function of pH (1.37 +/- 0.06 kcal mol-1 K-1), agrees with the value determined by differential scanning calorimetry. pH-dependent changes in the Soret region indicate that a group or groups in the heme environment of the denatured protein, probably 1 or both heme propionates, ionize with a pK near 4. The C102T variant exhibits both enthalpy and entropy convergence with a delta HD of 1.30 kcal mol-1 residue-1 at 373.6 K and a delta SD of 4.24 cal mol-1 K-1 residue-1 at 385.2 K. These values agree with those for other single-domain, globular proteins.     

NADPH binding induced proton ionization as a cause of nonlinear heat capacity changes in glutamate dehydrogenase

Functional group interactions involved in the formation of the glutamate dehydrogenase-NADPH binary complex have been studied by three independent but complementary approaches: the pH dependence of the overall dissociation constant measured by an improved differential spectroscopic technique; the pH dependence of the enthalpy of complex formation measured by flow calorimetry; and the pH dependence of the number of protons released to, or taken up from, the solvent in the complex formation reaction, measured by titration. We conclude that the coenzyme binds to the enzyme through three distinguishable interactions: a pH-independent process involving the binding of the reduced nicotinamide ring; a relatively weak "proton-stabilizing" process, occurring at low pH involving the shift at a pK of 6.3 in the free enzyme to 7.0 in the enzyme-NADPH complex; and a stronger "proton-destabilizing" process, occurring at a higher pH involving a shift of a pK of 8.5 in the enzyme down to 6.9 in the enzyme-NADPH complex. The proton ionization of the free enzyme involved in this third interaction exhibits some unusual thermodynamic parameters, having delta Go = +11.5 +/- 0.1 kcal mol-1, delta Ho = +19 +/- 1 kcal mol-1, and delta So = +23 eu. We show here that this proton ionization step is directly related to and indeed constitutes the "implicit" shift in enzyme macrostates which we have shown to be responsible for the existence of large highly nonlinear delta Cpo effects in the formation of this complex [Fisher, H. F., Colen, A. H., & Medary, R. T. (1981) Nature (London) 292, 271-272].     

Titration of the phase transition of phosphatidylserine bilayer membranes. Effects of pH, surface electrostatics, ion binding, and head-group hydration

The dependence of the gel-to-fluid phase transition temperature of dimyristoyl- and dipalmitoylphosphatidylserine bilayers on pH, NaCl concentration, and degree of hydration has been studied with differential scanning calorimetry and with spin-labels. On protonation of the carboxyl group (pK2app = 5.5), the transition temperature increases from 36 to 44 degrees C in the fully hydrated state of dimyristoylphosphatidylserine (from 54 to 62 degrees C for dipalmitoylphosphatidylserine), at ionic strength J = 0.1. In addition, at least two less hydrated states, differing progressively by 1 H2O/PS, are observed at low pH with transition temperatures of 48 and 52 degrees C for dimyristoyl- and 65 and 68.5 degrees C for dipalmitoylphosphatidylserine. On deprotonation of the amino group (pK3app = 11.55) the transition temperature decreases to approximately 15 degrees C for dimyristoyl- and 32 degrees C for dipalmitoylphosphatidylserine, and a pretransition is observed at approximately 6 degrees C (dimyristoylphosphatidylserine) and 21.5 degrees C (dipalmitoylphosphatidylserine), at J = 0.1. No titration of the transition is observed for the fully hydrated phosphate group down to pH less than or equal to 0.5, but it affinity for water binding decreases steeply at pH greater than or equal to 2.6. Increasing the NaCl concentration from 0.1 to 2.0 M increases the transition temperature of dimyristoyphosphatidylserine by approximately 8 degrees C at pH 7, by approximately 5 degrees at pH 13, and by approximately 0 degrees C at pH 1. These increases are attributed to the screening of the electrostatic titration-induced shifts in transition temperature. On a further increase of the NaCl concentration to 5.5 M, the transition temperature increases by an additional 9 degree C at pH 7, 13 degree C at pH 13, approximately 7 degree C in the fully hydrated state at pH 1, and approximately 4 and approximately 0 degree C in the two less hydrated states. These shifts are attributed to displacement of water of hydration by ion binding. From the salt dependence it is deduced that the transition temperature shift at the carboxyl titration can be accounted for completely by the surface charge and change in hydration of approximately 1 H2O/lipid, whereas that of the amino group titration arises mostly from other sources, probably hydrogen bonding. The shifts in pK (delta pK2 = 2.85, delta pK3 = 1.56) are consistent with a reduced polarity in the head-group region, corresponding to an effective dielectric constant epsilon approximately or equal to 30, together with surface potentials of psi congruent to -100 and -150 mV at the carboxyl and amino group pKs, respectively. The transition temperature of dimyristoylphosphatidylserine-water mixtures decreases by approximately 4 degree C each water/lipid molecule added, reaching a limiting value at a water content of approximately 9-10 H2O/lipid molecule.     

Thermodynamic parameters of cytochrome c3-ferredoxin complex formation

The complex formation between cytochrome c3 and ferredoxin I from Desulfovibrio desulfuricans Norway was studied by microcalorimetric and pH-stat titration measurements. The stoichiometry of the complex was found to be one molecule of cytochrome c3 per monomer of ferredoxin I. The association constant determined at T = 283 K in tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer, 10(-2) M and pH 7.7, was KA = 1.3 X 10(6) M-1. Though the enthalpy (delta H = 19 +/- 1 kJ.mol-1) and the entropy (delta S = 183 J.K-1.mol-1) were positive and consistent with a hydrophobic process involved in the interaction, the analysis of ionic strength dependence exhibited an important electrostatic effect on the association. The use of both Tris-HCl and phosphate buffers during microcalorimetric experiments showed proton release at pH 6.6. The pH-stat study of proton release indicated that one of the charged groups involved in the interacting site underwent a pK shift from 7.35 to 6.05.     

An investigation of the equilibria between aqueous ribose, ribulose, and arabinose

The thermodynamics of the equilibria between aqueous ribose, ribulose, and arabinose were investigated using high-pressure liquid chromatography and microcalorimetry. The reactions were carried out in aqueous phosphate buffer over the pH range 6.8-7.4 and over the temperature range 313.15-343.75 K using solubilized glucose isomerase with either Mg(NO3)2 or MgSO4 as cofactors. The equilibrium constants (K) and the standard state Gibbs energy (delta G degrees) and enthalpy (delta H degrees) changes at 298.15 K for the three equilibria investigated were found to be: ribose(aq) = ribulose(aq) K = 0.317, delta G degrees = 2.85 +/- 0.14 kJ mol-1, delta H degrees = 11.0 +/- 1.5 kJ mol-1; ribose(aq) = arabinose(aq) K = 4.00, delta G degrees = -3.44 +/- 0.30 kJ mol-1, delta H degrees = -9.8 +/- 3.0 kJ mol-1; ribulose(aq) = arabinose(aq) K = 12.6, delta G degrees = -6.29 +/- 0.34 kJ mol-1, delta H degrees = -20.75 +/- 3.4 kJ mol-1. Information on rates of the above reactions was also obtained. The temperature dependencies of the equilibrium constants are conveniently expressed as R in K = -delta G degrees 298.15/298.15 + delta H degrees 298.15[(1/298.15)-(1/T)] where R is the gas constant (8.31441 J mol-1 K-1) and T the thermodynamic temperature.     

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.     

Acylation of subtilisin A by aryl esters: contribution to rate limitation by a physical step preceding general acid-base catalysis

The specificity ratios kc/Km = k for subtilisin A catalyzed hydrolysis of five aryl esters of N-(methoxycarbonyl)-L-Phe (McPhe) were determined at pH 7.03 and its pD equivalent. The ratios are independent of the electronic properties of the leaving group substituent. Kinetic solvent isotope effects, Dk, increase from about 0.9 to 1.3 as leaving group ability decreases from p-nitrophenolate to p-methoxyphenolate. The k of N-(methoxycarbonyl)-L-phenylalanine p-nitrophenyl ester (NPE) with native enzyme exhibits a strong temperature dependence; delta H* = 87 +/- 3 kJ mol-1 and delta S* = 148 +/- 14 J K-1 mol-1 at 25 degrees C (H2O). The Dk with this substrate is 1.36 at 13.6 degrees C, declines to 0.89 at 25 degrees C, and then increases to 1.04 at 39.4 degrees C. Above neutral pH(D), with McPhe NPE as substrate, the dependence of k is for the dissociated form of a single base of pKapp = 7.38 +/- 0.03 in H2O and 7.67 +/- 0.03 in D2O. The pKapp values are apparently those of the uncomplexed native protein. By contrast, k of 3-phenylpropanoic acid (Prop) p-nitrophenyl ester exhibits a weaker temperature dependence; delta H* = 20 kJ mol-1 and delta S* = -90 J K-1 mol-1 (H2O) at 25 degrees C. The Dk are larger than those for McPhe NPE, decreasing from 1.99 at 20.5 degrees C to 1.74 at 46.1 degrees C. These results, combined with those of previous studies, are consistent with limitation of k by at least two processes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Basis for the equilibrium constant in the interconversion of l-lysine and l-beta-lysine by lysine 2,3-aminomutase

l-beta-lysine and beta-glutamate are produced by the actions of lysine 2,3-aminomutase and glutamate 2,3-aminomutase, respectively. The pK(a) values have been titrimetrically measured and are for l-beta-lysine: pK(1)=3.25 (carboxyl), pK(2)=9.30 (beta-aminium), and pK(3)=10.5 (epsilon-aminium). For beta-glutamate the values are pK(1)=3.13 (carboxyl), pK(2)=3.73 (carboxyl), and pK(3)=10.1 (beta-aminium). The equilibrium constants for reactions of 2,3-aminomutases favor the beta-isomers. The pH and temperature dependencies of K(eq) have been measured for the reaction of lysine 2,3-aminomutase to determine the basis for preferential formation of beta-lysine. The value of K(eq) (8.5 at 37 degrees C) is independent of pH between pH 6 and pH 11; ruling out differences in pK-values as the basis for the equilibrium constant. The K(eq)-value is temperature-dependent and ranges from 10.9 at 4 degrees C to 6.8 at 65 degrees C. The linear van't Hoff plot shows the reaction to be enthalpy-driven, with DeltaH degrees =-1.4 kcal mol(-1) and DeltaS degrees =-0.25 cal deg(-1) mol(-1). Exothermicity is attributed to the greater strength of the bond C(beta)-N(beta) in l-beta-lysine than C(alpha)-N(alpha) in l-lysine, and this should hold for other amino acids.     

Determination of the redox properties of the Rieske [2Fe-2S] cluster of bovine heart bc1 complex by direct electrochemistry of a water-soluble fragment.

The redox potential of the Rieske [2Fe-2S] cluster of the bc1 complex from bovine heart mitochondria was determined by cyclic voltammetry of a water-soluble fragment of the iron/sulfur protein. At the nitric-acid-treated bare glassy-carbon electrode, the fragment gave an immediate and stable quasireversible response. The midpoint potential at pH 7.2, 25 degrees C and I of 0.01 M was Em = +312 +/- 3 mV. This value corresponds within 20 mV to results of an EPR-monitored dye-mediated redox titration. With increasing ionic strength, the midpoint potential decreased linearly with square root of I up to I = 2.5 M. From the cathodic-to-anodic peak separation, the heterogeneous rate constant, k degrees, was calculated to be approximately 2 x 10(-3) cm/s at low ionic strength; the rate constant increased with increasing ionic strength. From the temperature dependence of the midpoint potential, the standard reaction entropy was calculated as delta S degrees = -155 J.K-1.mol-1. The pH dependence of the midpoint potential was followed over pH 5.5-10. Above pH 7, redox-state-dependent pK changes were observed. The slope of the curve, -120 mV/pH above pH9, indicated two deprotonations of the oxidized protein. The pKa values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pKa,ox of approximately 7.5 could also be observed in the optical spectrum of the oxidized protein. Redox-dependent pK values of the iron/sulfur protein are considered to be essential for semiquinone oxidation at the Qo center of the bc1 complex.     

Thermodynamics of ion binding to phosphatidic acid bilayers. Titration calorimetry of the heat of dissociation of DMPA.

The heat of dissociation of the second proton of 1,2-dimyristoylphosphatidic acid (DMPA) was studied as a function of temperature using titration calorimetry. The dissociation of the second proton of DMPA was induced by addition of NaOH. From the calorimetric titration experiment, the intrinsic pK0 for the dissociation reaction could be determined by applying the Gouy-Chapman theory. pK0 decreases with temperature from ca. 6.2 at 11 degrees C to 5.4 at 54 degrees C. From the total heat of reaction, the dissociation enthalpy, delta Hdiss, was determined by subtracting the heat of neutralization of water and the heat of dilution of NaOH. In the temperature range between 2 and 23 degrees C, delta Hdiss is endothermic with an average value of ca. 2.5 kcal.mol-1 and shows no clear-cut temperature dependence. In the temperature range between 23 and 52 degrees C, delta Hdiss calculated after subtraction of the heat of neutralization and dilution is not the true dissociation enthalpy but includes contributions from the phase transition enthalpy, delta Htrans, as the pH jump induces a transition from the gel to the liquid-crystalline phase. The delta Cp for the reaction enthalpy observed in this temperature range is positive. Above 53 degrees C, the pH jump induces again only the dissociation of the second proton, and the bilayers stay in the liquid-crystalline phase. In this temperature range, delta Hdiss seems to decrease with temperature. The thermodynamic data from titration calorimetry and differential scanning calorimetry as a function of pH can be combined to construct a complete enthalpy-temperature diagram of DMPA in its two ionization states.     

Effects of pH, ionic strength, and temperature on activation by calmodulin an catalytic activity of myosin light chain kinase

The reversible association of Ca42+-calmodulin with the inactive catalytic subunit of myosin light chain kinase results in the formation of the catalytically active holoenzyme complex [Blumenthal, D. K., & Stull, J. T. (1980) Biochemistry 19, 5608--5614]. The present study was undertaken in order to determine the effects of pH, temperature, and ionic strength on the processes of activation and catalysis. The catalytic activity of myosin light chain kinase, when fully activated by calmodulin, exhibited a broad pH optimum (greater than 90% of maximal activity from pH 6.5 to pH 9.0), showed only a slight inhibition by moderate ionic strengths (less than 20% inhibition at mu = 0.22), and displayed a marked temperature dependence (Q10 congruent to 2; Ea = 10.4 kcal mol-1). Thermodynamic parameters calculated from Arrhenius plots indicate that the Gibb's energy barrier associated with the rate-limiting step of catalysis is primarily enthalpic. The process of kinase activation by calmodulin had a narrower pH optimum (pH 6.0--7.5) than did catalytic activity, was markedly inhibited by increasing ionic strength (greater than 70% inhibition at mu = 0.22), and exhibited nonlinear van't Hoff plots. Between 10 and 20 degrees C, activation was primarily entropically driven (delta S degrees congruent to 40 cal mol-1 deg-1; delta H degrees = -900 cal mol-1), but between 20 and 30 degrees C, enthalpic factors predominated in driving the activation process (delta S degrees congruent to 10 cal mol-1 deg-1; delta H degrees = -9980 cal mol-1). The apparent change in heat capacity (delta Cp) accompanying activation was estimated to be -910 cal mol-1 deg-1. On the basis of these data we propose that although hydrophobic interactions between calmodulin and the kinase are necessary for the activation of the enzyme, other types of interactions such as hydrogen bonding, ionic, and van der Waals interactions also make significant and probably obligatory contributions to the activation process.     

1H-nuclear magnetic resonance studies of the neuropeptide head activator

The 1H-NMR spectrum of the neuropeptide head activator in aqueous solution has been completely assigned by two-dimensional NMR spectroscopy and selective deuteration. The apparent pseudo-first-order exchange rate, kex, of the backbone amide protons and the correspondent activation enthalpies, delta H not equal to, were determined. The exchange rates decrease and the activation enthalpies increase from the N-terminal to the C-terminal part of the peptide. The exchange rates vary from 21 to 0.3 s-1 at 274 K, the activation enthalpies from 60 to 75 kJ.mol-1. The pK values of the terminal carboxyl group and of the lysine amino group have been estimated as 3.3 and 10.3, respectively. The NMR results are in line with a dimeric structure in an antisymmetric arrangement of the subunits, forming an antiparallel beta-pleated sheet between C-terminal segments. The peptide bonds between pGlu-1, Pro-2 and Pro-3 are predominantly in trans-configuration, in fact no cis-isomers can be observed spectroscopically. The structure appears to be very stable; in the temperature and pH range studied, i.e., from 274 to 338 K and from pH 0.8 to pH 11.6, there are no spectroscopic indications for a global structural change.     

The electrophoretic mobility of normal and leukaemic cells of mice

1. The pH-mobility relationships for saline-washed cells from a mouse strain of acute lymphoblastic leukaemia were examined before and after treatment with lower aldehydes, diazomethane and neuraminidase (EC 3.2.1.18). 2. The content of sialic acid released into the supernatant fluid of neuraminidase-treated cells was measured. 3. The stability of the charge-determining structures to temporary changes in environment (pH and ionic strength) was established. 4. Similar measurements were made on lymph-node cells obtained from non-leukaemic mice (a resistant and a leukaemia-susceptible strain were examined). 5. It is deduced that both the malignant and the non-malignant cell possess two dissociable acid functions at the cell surface, a carboxyl group of sialic acid and another acidic group(s), probably carboxyl, of pK 3.0-4.5. The malignant cells, however, have a basic dissociable function not present in the non-malignant types. 6. Suggestions are made as to how the difference in surface chemistry may be related to the problem of malignancy.     

DSC studies of the conformational stability of barstar wild-type.

The temperature induced unfolding of barstar wild-type of bacillus amyloliquefaciens (90 residues) has been characterized by differential scanning microcalorimetry. The process has been found to be reversible in the pH range from 6.4 to 8.3 in the absence of oxygen. It has been clearly shown by a ratio of delta HvH/delta Hcal near 1 that denaturation follows a two-state mechanism. For comparison, the C82A mutant was also studied. This mutant exhibits similar reversibility, but has a slightly lower transition temperature. The transition enthalpy of barstar wt (303 kJ mol-1) exceeds that of the C82A mutant (276 kJ mol-1) by approximately 10%. The heat capacity changes show a similar difference, delta Cp being 5.3 +/- 1 kJ mol-1 K-1 for the wild-type and 3.6 +/- 1 kJ mol-1 K-1 for the C82A mutant. The extrapolated stability parameters at 25 degrees C are delta G0 = 23.5 +/- 2 kJ mol-1 for barstar wt and delta G0 = 25.5 +/- 2 kJ mol-1 for the C82A mutant.     

Autoxidation of myoglobin from bigeye tuna fish (Thunnus obesus)

Native oxymyoglobin (MbO2) was isolated directly from the skeletal muscle of bigeye tuna (Thunnus obesus) with complete separation from metmyoglobin (metMb) on a CM-cellulose column. It was examined for its stability properties over a wide range of pH values (pH 5-12) in 0.1 M buffer at 25 degrees C. When compared with sperm whale MbO2 as a reference, the tuna MbO2 was found to be much more susceptible to autoxidation. Kinetic analysis has revealed that the rate constant for a nucleophilic displacement of O2- from MbO2 by an entering water molecule is 10-times higher than the corresponding value for sperm whale MbO2. The magnitude of the circular dichroism of bigeye tuna myoglobin at 222 nm was comparable to that of sperm whale myoglobin, but its hydropathy profile revealed the region corresponding to the distal side of the heme iron to be apparently less hydrophobic. The kinetic simulation also demonstrated that accessibility of the solvent water molecule to the heme pocket is clearly a key factor in the stability properties of the bound dioxygen.     

The beta-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme-catalysed reactions.

The effects of pH and temperature on Michaelis constant (Km) and maximum velocity (Vmax.) and of NaCl on the activity of the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase EC 3.2.1.21) from cultures of Botryodiplodia theobromae Pat. have been studied. 2. Donor binding and inhibition of activity by glucose were dependent on the ionization of a group (pK 6.0) that appeared to be an imidazole group. 3. Catalytic activity and the stimulation of activity by glycerol were dependent on the ionization of two groups, which appeared to be a carboxy group and an imidazole group. 4. The Arrhenius activation energy (Ea) calculated from results obtained at pH 4.0 and 5.0 was about 45--46kJ.mol-1. 5. The enthalpies (delta H0) calculated from results obtained at pH 4.0 and 5.0 were similar (about -4kJ.mol-1), whereas at pH 6.5 the value was about -33kJ.mol-1. 6. The entropies (delta S0) calculated from these results at 37 degrees C were -21, -22 and -118J.K-1.mol-1 at pH 4.0, 5.0 and 6.5 respectively. A low concentration of NaCl (16.6 mM) stimulated enzymic activity and decreased the Km for the donor, whereas high concentrations (up to 500 mM) inhibited enzymic activity, increased the Km and had no effect on Vmax. 8. Plots of initial velocity data obtained in the presence of dioxan as 1/v against the ratio of the molar concentration of dioxan to that of water were linear. 9. The results are discussed in terms of the enzyme mechanism.     

Role of globin moiety in the autoxidation reaction of oxymyoglobin: effect of 8 M urea.

It is in the ferrous form that myoglobin or hemoglobin can bind molecular oxygen reversibly and carry out its function. To understand the possible role of the globin moiety in stabilizing the FeO2 bond in these proteins, we examined the autoxidation rate of bovine heart oxymyoglobin (MbO2) to its ferric met-form (metMb) in the presence of 8 M urea at 25 degrees C and found that the rate was markedly enhanced above the normal autoxidation in buffer alone over the whole range of pH 5-13. Taking into account the concomitant process of unfolding of the protein in 8 M urea, we then formulated a kinetic procedure to estimate the autoxidation rate of the unfolded form of MbO2 that might appear transiently in the possible pathway of denaturation. As a result, the fully denatured MbO2 was disclosed to be extremely susceptible to autoxidation with an almost constant rate over a wide range of pH 5-11. At pH 8.5, for instance, its rate was nearly 1000 times higher than the corresponding value of native MbO2. These findings lead us to conclude that the unfolding of the globin moiety allows much easier attack of the solvent water molecule or hydroxyl ion on the FeO2 center and causes a very rapid formation of the ferric met-species by the nucleophilic displacement mechanism. In the molecular evolution from simple ferrous complexes to myoglobin and hemoglobin molecules, therefore, the protein matrix can be depicted as a breakwater of the FeO2 bonding against protic, aqueous solvents.