Thermodynamic study of protein dynamic structure in the oxygen binding reaction of myoglobin

We examined the flash photolysis of oxy complexes of sperm whale myoglobin (Mb) on the nanosecond time scale at ambient temperatures. In this time range, we can observe the geminate reaction of Mb with the O2 ligand existing in the protein matrix after the photodissociation from the heme iron. We found that the fraction of the geminate component to the total O2 photodissociation exhibited temperature dependences. The geminate fraction decreased with rising temperature, indicating that the protein fluctuation is enhanced at high temperature because of thermal agitation. However, the temperature-dependent behavior showed a break at 20 degrees C. Concerning the geminate O2 escaping reaction from the protein matrix to the solvent region, the activation energy above 20 degrees C (0.4 +/- 0.4 kcal/mol) is significantly lower than that below 20 degrees C (5.1 +/- 0.4 kcal/mol). Thermodynamic analysis on the basis of the transition state theory indicated that the O2 escaping reaction above 20 degrees C is entropy dominated whereas that below 20 degrees C is enthalpy dominated. The results were qualitatively compatible with the theoretical prediction by J. Kottalam and D. A. Case [1988) J. Am. Chem. Soc. 110, 7690-7697). Comparing the kinetic and thermodynamic process of the O2 geminate reaction among several Mbs, we concluded that the geminate O2 reaction with Mb is governed by the dynamic motion of the protein which is sensitively controlled by the static interaction of the heme moiety with the surroundings.     

Kinetic, structural, and spectroscopic identification of geminate states of myoglobin: a ligand binding site on the reaction pathway

Elementary steps or geminate states in the reaction of gaseous ligands with transport proteins delineate the trajectory of the ligand and its rebinding to the heme. By use of kinetic studies of the 765-nm optical "conformation" band, three geminate states were identified for temperatures less than approximately 100 K. MbCO, which is accumulated by photolysis between 1.2 and approximately 10 K, was characterized by our previous optical and X-ray absorption studies [Chance, B., Fischetti, R., & Powers, L. (1983) Biochemistry 22, 3820-3829]. Between 10 and approximately 100 K, geminate states that are also identified that have recombination rates of approximately 10(3) s-1 and approximately 10(-5) s-1 (40 K). Thus, it is possible to maintain a steady-state nearly homogeneous population of the slowest recombining geminate state, Mb, by regulated continuous illumination (optical pumping). Both X-ray absorption and resonance Raman studies under similar conditions of optical pumping show that the heme structure around the iron in Mb is similar to that of MbCO. In both geminate states, the iron-proximal histidine distance remains unchanged (+/- 0.02 A) from that of MbCO while the iron to pyrrole nitrogen average distance has not fully relaxed to that of the deoxy state. In MbCO the CO remains close to iron but not bound, and the Fe...CO angle, which is bent in MbCO (127 +/- 4 degrees C), is decreased by approximately 15 degrees [Powers, L., Sessler, J. L., Woolery, G. L., & Chance, B. (1984) Biochemistry 23, 5519-5523]. The CO molecule in Mb, however, has moved approximately 0.7 A further from iron. Computer graphics modeling of the crystal structure of MbCO places the CO in a crevice in the heme pocket that is just large enough for the CO molecule end-on. Above approximately 100 K resonance Raman studies show that this structure relaxes to the deoxy state.     

A kinetic description of ligand binding to sperm whale myoglobin

Nanosecond recombination time courses were measured by photolyzing O2, NO, CO, methyl, ethyl, n-propyl, n-butyl, and tert-butyl isocyanide complexes of sperm whale myoglobin with a 30-ns laser pulse at pH 7, 20 degrees C. Absorbance was measured both during and after the excitation pulse and as a function of laser light intensity. The results were analyzed quantitatively in terms of a three-step reaction scheme, MbX in equilibrium B in equilibrium C in equilibrium Mb + X, where Mb is myoglobin, B represents a geminate state in which the ligand is present in the distal pocket but not covalently bound to the iron atom, and C, a state in which the ligand is still embedded in the protein but further away from the heme group. The fitted rate parameters were required to be consistent with the observed overall quantum yield, Q, which had been measured independently using much longer (approximately 0.5 ms) xenon flash pulses. Three major conclusions were derived from these analyses. First, the overall quantum yield of the ligand complex is determined primarily by the competition between the rate of iron-ligand bond formation from the initial photoproduct, kB----MbX, and the rate of migration away from state B, kB----C. For example, kB----C approximately equal to 30-100 microseconds-1 for all three gaseous ligands, whereas both Q and kB----MbX vary over 3 orders of magnitude (i.e. NO, Q = 0.001, kB----MbX approximately equal to 16,000 microseconds-1; O2, Q = 0.1, kB----MbX approximately equal to 500 microseconds-1; CO, Q = 1.0, kB----MbX approximately equal to 2 microseconds-1). Second, for NO, O2, and the isonitriles, the rate-limiting step in the overall association reaction starting from ligand in solution is the formation of state B. The rate constant for this process varies from 2 X 10(7) M-1 s-1 for the gaseous ligands to 0.02-1.4 X 10(5) M-1 s-1 for the isonitriles. In contrast, the B to MbX transition is limiting for CO binding. Third, for all the ligands except CO, the overall rate of dissociation is limited significantly both by the rate of thermal bond disruption, kMbX----B, and the competition between geminate recombination and migration away from the distal pocket (i.e. kB----C/(kB----MbX + kB----C]. In the case of CO, the rate of bond disruption is equal to the observed dissociation rate constant.     

The temperature-dependence of adenylate cyclase from baker''s yeast.

The Michaelis constant of membrane-bound adenylate cyclase increased from 1.1 to 1.8 mM between 7 and 38 degrees C (delta H = 13 kJ/mol). Over this temperature range, the maximum velocity increased 10-fold, and the Arrhenius plot was nearly linear, with an average delta H* of 51 kJ/mol. The temperature-dependence of the reaction rate at 2 mM-ATP was examined in more detail: for Lubrol-dispersed enzyme, Arrhenius plots were nearly linear with average delta H* values of 45 and 68 kJ/mol, respectively, for untreated and gel-filtered enzymes; for membrane-bound enzyme, delta H changed from 40 kJ/mol above about 21 degrees C to 62 kJ/mol below 21 degrees C, but this behaviour does not necessarily indicate an abrupt, lipid-induced, transition in the reaction mechanism.     

Stabilities of consecutive A.C, C.C, G.G, U.C, and U.U mismatches in RNA internal loops: Evidence for stable hydrogen-bonded U.U and C.C.+ pairs.

The stability and structure of RNA duplexes with consecutive A.C, C.A, C.C, G.G, U.C, C.U, and U.U mismatches were studied by UV melting, CD, and NMR. The results are compared to previous results for GA and AA internal loops [SantaLucia, J., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819; Peritz, A., Kierzek, R., & Turner, D.H. (1991) Biochemistry 30, 6428-6436)]. The observed order for stability increments of internal loop formation at pH 7 is AG = GA approximately UU greater than GG greater than or equal to CA greater than or equal to AA = CU = UC greater than or equal to CC greater than or equal to AC. The results suggest two classes for internal loops with consecutive mismatches: (1) loops that stabilize duplexes and have strong hydrogen bonding and (2) loops that destabilize duplexes and may not have strong hydrogen bonding. Surprisingly, rCGCUUGCG forms a very stable duplex at pH 7 in 1 M NaCl with a TM of 44.8 degrees C at 1 x 10(-4) M and a delta G degrees 37 of -7.2 kcal/mol. NOE studies of the imino protons indicate hydrogen bonding within the U.U mismatches in a wobble-type structure. Resonances corresponding to the hydrogen-bonded uridines are located at 11.3 and 10.4 ppm. At neutral pH, rCGCCCGCG is one of the least stable duplexes with a TM of 33.2 degrees C and delta G degrees 37 of -5.1 kcal/mol. Upon lowering the pH to 5.5, however, the TM increases by 12 degrees C, and delta G degrees 37 becomes more favorable by 2.5 kcal/mol. The pH dependence of rCGCCCGCG may be due to protonation of the internal loop C's, since no changes in thermodynamic parameters are observed for rCGCUUGCG between pH 7 and 5.5. Furthermore, two broad imino proton resonances are observed at 10.85 and 10.05 ppm for rCGCCCGCG at pH 5.3, but not at pH 6.5. This is also consistent with C.C+ base pairs forming at pH 5.5. rCGCCAGCG and rGGCACGCC have a small pH dependence, with TM increases of 5 and 3 degrees C, respectively, upon lowering the pH from 7 to 5.5. rCGCCUGCG and rCGCUCGCG also show little pH dependence, with TM increases of 0.8 and 1.4 degrees C, respectively, upon lowering the pH to 5.5.(ABSTRACT TRUNCATED AT 400 WORDS)     

A time-resolved photoacoustic calorimetry study of the dynamics of enthalpy and volume changes produced in the photodissociation of carbon monoxide from sperm whale carboxymyoglobin

The dynamics of the enthalpy and volume changes produced in the photodissociation of carbon monoxide from sperm whale myoglobin is investigated by time-resolved photoacoustic calorimetry. The enthalpy and volume changes for the formation of the geminate pair, which occurs within 50 ns of photolysis, are delta H = -2.2 +/- 2.8 kcal/mol and delta V = -10.0 +/- 1.0 mL/mol relative to carboxymyoglobin. The enthalpy and volume changes associated with formation of deoxymyoglobin and solvated carbon monoxide, formed with a half-life of 702 +/- 31 ns at 20 degrees C, are delta H = 14.6 +/- 3.4 kcal/mol and delta V = 5.8 +/- 1.0 mL/mol relative to carboxymyoglobin.     

Isolation and properties of myoglobin from murine skeletal muscle

Myoglobin (Mb) was isolated from skeletal muscle of JCL-ICR mice by heat denaturation-gel filtration combined with ion exchange chromatography or chromatofocusing by which isoelectric point of the main component was estimated as 7.63 +/- 0.09 (20 degrees C). The Mb was homogeneous by gel electrophoretic and ultracentrifugal analysis. The molecular weight by sedimentation equilibrium was 1.80 X 10(4) and essentially identical with the values by the iron analysis (1.82 X 10(4)) and amino acid composition (1.78 X 10(4)) in which one residue of cysteine was found per molecule. The spectroscopic properties of deoxy-, oxy-, carboxy- and ferri-derivatives of the protein were determined in ultraviolet, Soret and visible regions. The pK' of acid-alkaline transition of the ferri-form was estimated as 8.57 +/- 0.30 (30 degrees C) from the pH-dependent spectral changes. The oxygen equilibrium studies revealed complete absence of such allosteric properties as heme-heme interaction, anion effect and Bohr effect. Oxygen tension for the half-oxygenation (P50) was 0.69 +/- 0.06 Torr (20 degrees C) and its temperature-dependent change gave the delta H degrees of -14.1 kcal/mole.     

Proximal and distal influences on ligand binding kinetics in microperoxidase and heme model compounds

We use laser flash photolysis and time-resolved Raman spectroscopy of CO-bound heme complexes to study proximal and distal influences on ligand rebinding kinetics. We report kinetics of CO rebinding to microperoxidase (MP) and 2-methylimidazole ligated Fe protoporphyrin IX in the 10 ns to 10 ms time window. We also report CO rebinding kinetics of MP in the 150 fs to 140 ps time window. For dilute, micelle-encapsulated (monodisperse) samples of MP, we do not observe the large amplitude geminate decay at approximately 100 ps previously reported in time-resolved IR measurements on highly concentrated samples [Lim, M., Jackson, T. A., and Anfinrud, P. A. (1997) J. Biol. Inorg. Chem. 2, 531-536]. However, for high concentration aggregated samples, we do observe the large amplitude picosecond CO geminate rebinding and find that it is correlated with the absence of the iron-histidine vibrational mode in the time-resolved Raman spectrum. On the basis of these results, the energetic significance of a putative distal pocket CO docking site proposed by Lim et al. may need to be reconsidered. Finally, when high concentration samples of native myoglobin (Mb) were studied as a control, an analogous increase in the geminate rebinding kinetics was not observed. This verifies that studies of Mb under dilute conditions are applicable to the more concentrated regime found in the cellular milieu.     

Viscosity-dependent energy barriers and equilibrium conformational fluctuations in oxygen recombination with hemerythrin

The recombination kinetics of photo-dissociated oxyhemerythrin (Sipunculus nudus) have been investigated between 298 K and 90 K. Fast geminate recombinations compete with oxygen escape into the solvent, from which a subsequent slower bimolecular rebinding takes place. In phosphate buffer (pH 7.7) at 278 K, the fast and slow processes are exponential and have comparable amplitudes. Whereas the oxygen escape rate rapidly decreases upon increasing the viscosity, the inward rate from the solvent is found to be independent of viscosity, up to about 50 cP (50 mPa.s). The data suggest that a Brownian-motion-driven displacement of one or several side-chain residues is implied in oxygen escape from within the protein and also that hemerythrin undergoes a conformational change in the deoxy state. At higher viscosities and lower temperature only the geminate phase is observed and the kinetics progressively depart from an exponential. Below about 130 K, the kinetics resemble those reported in the literature for heme proteins. They are consistent with a temperature-independent non-equilibrium frozen distribution of conformational substates. However, between 190 K and 130 K, the profile of the kinetics is invariant on a log/log plot and the results simply differ by a translation along the log t axis. It is shown that this property is expected only for a temperature-dependent distribution of substates in a Boltzmann equilibrium. From room temperature, where rebinding is exponential, down to the 'freezing' temperature, the geminate recombinations display a variety of kinetic laws. It can be shown, however, that for a broad class of substate distributions, the initial slope of the kinetic plot follows an Arrhenius relationship. The activation energy is equal to that of the exponential rate constant measured at high temperature. This result establishes the conditions under which protein data obtained from low-temperature kinetics can be extrapolated to physiological temperature.     

A jump in an Arrhenius plot can be the consequence of a phase transition. The binding of ATP to myosin subfragment 1

The temperature dependence of the kinetics of the binding of ATP to myosin subfragment-1 was studied by an ATP chase technique in a rapid-flow-quench apparatus: (formula; see text) A temperature range of 30 degrees C to -15 degrees C was obtained with ethylene glycol as antifreeze. The Arrhenius plot of k2 is discontinuous with a jump at 12 degrees C. Above the jump delta H+ = 9.5 kcal/mol, below delta H+ = 28.5 kcal/mol. Few such Arrhenius plots are recorded in the literature but they are predicted from theory. Thus, we explain our results as a phase change of the subfragment 1-ATP system at 12 degrees C. This is in agreement with certain structural studies.     

Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea.

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.     

Water penetration and binding to ferric myoglobin

Flash photolysis investigations of horse heart metmyoglobin bound with NO (Mb(3+)NO) reveal the kinetics of water entry and binding to the heme iron. Photodissociation of NO leaves the sample in the dehydrated Mb(3+) (5-coordinate) state. After NO photolysis and escape, a water molecule enters the heme pocket and binds to the heme iron, forming the 6-coordinate aquometMb state (Mb(3+)H2O). At longer times, NO displaces the H2O ligand to reestablish equilibrium. At 293 K, we determine a value k(w) approximately 5.7 x 10(6) s(-1) for the rate of H2O binding and estimate the H2O dissociation constant as 60 mM. The Arrhenius barrier height H(w) = 42 +/- 3 kJ/mol determined for H2O binding is identical to the barrier for CO escape after photolysis of Mb(2+)CO, within experimental uncertainty, consistent with a common mechanism for entry and exit of small molecules from the heme pocket. We propose that both processes are gated by displacement of His-64 from the heme pocket. We also observe that the bimolecular NO rebinding rate is enhanced by 3 orders of magnitude both for the H64L mutant, which does not bind water, and for the H64G mutant, where the bound water is no longer stabilized by hydrogen bonding with His-64. These results emphasize the importance of the hydrogen bond in stabilizing H2O binding and thus preventing NO scavenging by ferric heme proteins at physiological NO concentrations.     

A test of the linear extrapolation of unfolding free energy changes over an extended denaturant concentration range.

Guanidine hydrochloride (GdnHCl) and thermally induced unfolding measurements on the oxidized form of Escherichia coli thioredoxin at pH 7 were combined for the purpose of assessing the functional dependence of unfolding free energy changes on denaturant concentration over an extended GdnHCl concentration range. Conventional analysis of GdnHCl unfolding exhibits a linear plot of unfolding delta G vs [GdnHCl] in the transition zone. In order to extend unfolding delta G measurements outside of that narrow concentration range, thermal unfolding measurements were performed using differential scanning calorimetry (DSC) in the presence of low to moderate concentrations of GdnHCl. The unfolding delta G values from the DSC measurements were corrected to 25 degrees C using the Gibbs-Helmholtz equation and mapped onto the delta G vs [GdnHCl] plot. The dependence of unfolding delta G on [GdnHCl] was found to be linear over the full denaturant concentration range, provided that the chloride ion concentration was kept at a threshold of greater than or equal to 1.5 M. In the DSC experiments performed in the presence of GdnHCl, chloride concentrations were maintained at 1.5 M by addition of appropriate amounts of NaCl. The linear extrapolation method (LEM) gives an unfolding free energy change in the absence of denaturant (delta G degrees N-U) in excellent agreement with the delta G determined by DSC measurement in 1.5 M NaCl. The various methods give a consensus unfolding delta G value of 8.0 kcal/mol at 25 degrees C in the absence of denaturant.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR study of Galeorhinus japonicus myoglobin. 1H-NMR evidence for a structural alteration on the active site of G. japonicus myoglobin upon azide ion binding

The heme molecular structure of the met-azido form of the myoglobin from the shark Galeorhinus japonicus has been investigated by 1H NMR. A nuclear Overhauser effect (NOE) was clearly observed among the heme peripheral side-chain proton signals of this complex, which undergoes thermal spin equilibrium between high-spin (S = 5/2) and low-spin (S = 1/2) states, and the NOE connectivities provided the assignment of the resonances from the heme C13(1)H2 and C17(1)H2 protons. Chemical shift inequivalence of these proton resonances not only provided information about the orientation of these methylene protons with respect to the heme plane, but also allowed characterization of the time-dependent build-up of the NOE between them, which yields the correlation time for the internal motion of the inter-proton vector. The relatively large mobility found for the C17(1)H2 group suggests that the carboxyl oxygen of the heme C17 propionate is not anchored to the apo-protein by a salt bridge. It has been shown that the ferric high-spin form of G. japonicus Mb possesses a penta-coordinated heme [Suzuki, T. (1987) Biochim. Biophys. Acta 914, 170-176; Yamamoto, Y., Osawa, A., Inoue, Y., Ch?j?, R. & Suzuki, T. (1990) Eur. J. Biochem. 192, 225-229] and that the conformation of both heme propionate groups is fixed with respect to the heme, as well as the apo-protein, by a salt bridge [Yamamoto, Y., Inoue, Y., Ch?j?, R. & Suzuki, T. (1990) Eur. J. Biochem. 189, 567-573]. Therefore the weakening or interruption of the interaction between the C17 propionate and His FG3 upon the changes of the coordination and spin state of the heme iron, during azide ion binding to ferric high-spin G. japonicus Mb, is attributed to the displacement of the FG corner of the apoprotein away from the heme C17 propionate group. A similar structural alteration has been revealed by X-ray structural analyses of unliganded and liganded forms of ferrous hemoproteins [Baldwin, J. & Chothia, C. (1979) J. Mol. Biol. 129, 175-220; Phillips, S.E.V. (1980) J. Mol. Biol. 142, 531-554].     

Physico-chemical aspects of solubility of myosin in aqueous media

Solubility of fish (Labio rohita) myosin has been studied at varying temperatures in presence of various inorganic salts like NaCl, KCl, NaBr, Na2SO4, KI, and organic solutes like sucrose and urea. The effect of pH on the solubility has also been studied both in absence and presence of NaCl. Thermal denaturation temperatures of myosin in presence of NaCl, KCl, NaBr and Na2SO4 were found to be 40 degrees, 40 degrees, 45 degrees and 50 degrees C respectively. Thermodynamic parameters like changes in standard free energy (delta G degrees), enthalpy (delta H degrees) and entropy (delta S degrees) for precipitation of myosin from solution phase to gel phase have been evaluated and the physico-chemical aspects have been critically discussed. The average delta G degrees for gel formation varied only between -30 and -40 kJ/mole of myosin, although the nature of solutes, temperature and folding state of protein have been grossly altered. A compensation effect has also been exhibited from the linear plot of average values of delta H degrees against T delta S degrees for various solutes.     

Effects of magnesium sulfate on an unfolding step of human cyanomet myoglobin.

The effects of magnesium sulfate (MgSO4) on an unfolding step of human cyanomet myoglobin (Mb) were examined for wild-type and three L-->A mutant Mbs. The unfolding was induced at acidic pH (3.6-4.5) with various concentrations of MgSO4 (0-2 M). The monophasic process was monitored by visible absorption spectroscopy. We observed quite nonlinear delta G not equal to-[MgSO4] relations for all the Mbs. delta G not equal to-[MgCl2] relations were also determined for a comparative study. Thermodynamic evaluation of the results indicated that an upward reflection of delta G not equal to-[MgSO4] relations in high [MgSO4] is caused by the strong Hofmeister effect of the salt. Results obtained for three mutants (L29A, L72A, and L104A) at pH 4.0 and 4.5 were consistent with our previous observation that the structure of the transition state is determined by the stability of Mb cores in the balance with the pH conditions of unfolding (T. Konno and I. Morishima. 1993. Biochim. Biophys. Acta. 1162:93-98).     

Investigations of photolysis and rebinding kinetics in myoglobin using proximal ligand replacements

We use laser flash photolysis and time-resolved Raman spectroscopy of CO-bound H93G myoglobin (Mb) mutants to study the influence of the proximal ligand on the CO rebinding kinetics. In H93G mutants, where the proximal linkage with the protein is eliminated and the heme can bind exogenous ligands (e.g., imidazole, 4-bromoimidazole, pyridine, or dibromopyridine), we observe significant effects on the CO rebinding kinetics in the 10 ns to 10 ms time window. Resonance Raman spectra of the various H93G Mb complexes are also presented to aid in the interpretation of the kinetic results. For CO-bound H93G(dibromopyridine), we observe a rapid large-amplitude geminate phase with a fundamental CO rebinding rate that is approximately 45 times faster than for wild-type MbCO at 293 K. The absence of an iron proximal ligand vibrational mode in the 10 ns photoproduct Raman spectrum of CO-bound H93G(dibromopyridine) supports the hypothesis that proximal ligation has a significant influence on the kinetics of diatomic ligand binding to the heme.     

Changes in the apparent quantum efficiency for photolysis of Hb(CO)1.

Recent studies suggest that the allosteric state of the protein surrounding the hemes in hemoglobin affects both geminate recombination of CO and the apparent quantum efficiency (AQE) for photolysis (Rohlfs, R.J., J.S. Olson, and Q.H. Gibson, 1988, J. Biol. Chem. 263: 1803-1813. We report combined flow/flash experiments in which the AQE for photolysis of Hb(CO)1 was measured as a function of time delay after its formation. Experiments were carried out at 20 degrees C in 0.1 M phosphate buffer at pH 7.0 with CO saturations of 10% or less. The AQE was observed to decrease from a value close to 1.0 at short times to approximately 0.6 after 2 s. The fundamental photolysis step for carboxyhemoglobin is known to have a quantum efficiency of nearly 1.0, whereas the lower AQE values we observe result from competition between rapid geminate recombination and a rapid reaction step leading to escape of the CO to the solution phase. Changes in AQE values reflect changes in these rapid reaction steps which presumably result from conformational change in Hb(CO)1. The change in AQE is consistent with conversion of one or more hemes to an R-like state but these changes could not be even approximately described in terms of a simple two-state allosteric model.     

Alteration of the proximal bond energy in the unliganded form of the homodimeric myoglobin from Nassa mutabilis. Kinetic and spectroscopic evidence.

CO binding kinetics to the homodimeric myoglobin (Mb) from Nassa mutabilis has been investigated between pH 1.9 and 7.0. Protonation of the proximal imidazole at low pH (less than or equal to 3.0) and the consequent cleavage of the HisF8NE2-Fe proximal bond brings about a approximately 20-fold increase of the second-order rate constant for CO binding. This process displays a pKa = 4.0 +/- 0.2, significantly higher than that observed in all other deoxygenated hemoproteins investigated up to now. Such a feature underlies a decreased energy for the HisF8NE2-Fe proximal bond in the unliganded form and it also appears supported by resonance Raman spectroscopy in the low frequency region of the Fe(II) deoxygenated hemoprotein. Further, the pH-rate profile of N. mutabilis Mb, like that of the homodimeric hemoglobin (Hb) from Scapharca inaequivalvis (Coletta, M., Boffi, A., Ascenzi, P., Brunori, M. and Chiancone, E. (1990) J. Biol. Chem. 265, 4828-4830), can be described only by assuming a concerted proton-linked transition with n = 1.8 +/- 0.1. Such a characteristic suggests, also on the basis of the amino acid sequence homology between N. mutabilis Mb and S. inaequivalvis Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different from that operative in other hemoproteins.