The acid-alkaline transition of a sea turtle myoglobin: coexistence at high pH of high- and low-spin forms

The microenvironment of the iron in a sea turtle Dermochelys coriacea myoglobin is studied using the spectroscopic techniques EPR and optical absorption. Optical absorption spectra in the visible region suggest a great homology between turtle Mb and other myoglobins, such as those from whale, human and elephant. The pK of the acid-alkaline transition is 8.4 slightly lower than the pK of whale and equal to that of elephant myoglobin. The EPR spectrum at pH 7.0 is characteristic of a high-spin configuration with axial symmetry (gx = gy = 5.95). At higher pH, this signal changes in a way different from that observed for whale myoglobin. We observe for turtle Mb both the formation of a low-spin configuration with rhombic symmetry (gx = 2.56, gy = 2.20, gz = 1.90) and of a high-spin species with rhombic distortion (gx = 6.79, gy = 5.18, gz = 2.12). This suggests a lowering of symmetry at the haem, so that now the x and y directions are no more equivalent. This can be explained by amino acid substitution at the distal positions of haem or to off-axial positioning of distal residues. The coexistence at high pH (pH 11.0) of these two spin forms could be explained by the existence of two protein conformations, in which the crystal field splitting factor, delta, and the electron exchange energy are of the same order, allowing the presence of different configurations simultaneously. The presence of different kinds of haem is ruled out by the experiments with nitrosyl turtle Mb and turtle Mb-F showing spectra very similar to those of whale myoglobin. The pk of the acid-alkaline transition, 8.5, obtained from EPR spectra, agrees very well with results from optical absorption.     

The mechanism of reaction of nitrosyl with met- and oxymyoglobin: an ESR study

In this ESR work we have studied the pentacoordinate symmetry in horse, whale and sperm-whale myoglobin (Mb) in different physical states such as solution and powder. Experiments were performed in which the following parameters were varied: the sample temperature, pH, reaction time with NO, and NO concentration. The results enabled us to explain the NO reaction mechanism in the oxy and met forms of myoglobin. The study of powder samples at different degrees of hydration allowed us to identify the diamagnetic intermediate species existent in the reaction of NO with met-Mb proposed in the literature. The results presented explain adequately the pH effect and temperature dependence observed in the ESR spectra obtained using the met-Mb sample solutions from Sigma Chemical Co., which consist of a mixture (13%) of Mb-O2.     

Electronic and stereochemical characterizations of the photoinduced intermediates of nitrosyl complexes of metal (S = 5/2)-substituted hemoproteins trapped at low temperature

Low temperature photolysis of nitric oxide from the nitrosyl complexes of ferric myoglobin (NO-Fe(III)Mb) and manganese(II)-porphyrin-substituted myoglobin (NO-Mn(II)Mb) was examined by electron paramagnetic resonance (EPR) spectroscopy in order to elucidate the electronic and structural natures of the photoinduced intermediates of these hemoprotein-ligand complexes trapped at low temperature. The photoproduct of NO-Fe(III)Mb at 5 K exhibited entirely new X-band EPR absorptions in the magnetic field strength from 0 to 0.4 tesla. The widespread absorption together with distinct, sharp zero-field absorption was consistently observed in the photoproduct of the isoelectronic NO-Mn(II)Mb. These novel ERP signals indicate a spin-coupled pair with an effective spin of S = 2 between the high spin metal center (S = 5/2) and the photodissociated NO (S = 1/2) trapped adjacent to the metal center. On the other hand, the photolyzed form of nitrosyl complexes of Fe(III)- and Mn(II)-Glycera hemoglobins, in which the distal histidine of Mb is replaced by a leucyl residue, exhibited somewhat broader EPR absorptions similar to those of the corresponding native Fe(III)- or unliganded Mn(II)-Glycera hemoglobins, respectively, indicating that the photodissociated NO molecule moved farther away from the metal center in the heme pocket. These observations show the importance of the interaction of the distal residue with the ligand in determining the nature of the photolyzed states.     

Optical spectroscopic observation of a metastable form of sperm whale myoglobin generated by reconstitution

The optical spectrum of Sperm Whale myoglobin, which has been freshly reconstituted with iron protoporphyrin-IX, is shown to be different from that obtained from the native myoglobin, and from the reconstituted, incubated myoglobin (These last two have equivalent absorption spectra). The effect is immediately evident as a shift of about +1 nm in the Soret band during incubation of freshly reconstituted metMb. Difference spectroscopy can be used to deconvolute changes in optical spectra occurring during and after Mb reconstitution into two components. The initial phase reflects incorporation of hemin into the protein matrix; this is already known to produce two forms, differing by relative hemin orientation. The rate of the second process follows the known pH dependence of iron protoporphyrin-IX reorientation. Presence of the second process indicates that the absorption spectrum of each of the two hemin-insertion Mb forms is unique, so interconversion between the two forms is monitored. Thus iron protoporphyrin-IX reorientation in proteins may be studied by visible spectroscopy.     

Investigations of optical line shapes and kinetic hole burning in myoglobin.

We present the results of an extensive investigation of the optical line shapes of deoxymyoglobin (Mb), the ligand-bound form (MbCO), and the low-temperature photoproduct (Mb*). The thermal properties and the pH dependence of the Soret band and the near infrared band III (approximately 760 nm) are analyzed, taking into account the underlying vibrational properties of the absorption bands. The strong temperature dependence associated with the Soret band of MbCO and band III of Mb indicates significant coupling to low-frequency modes that may not be directly observed in the resonance Raman spectra. On the basis of analogous line-shape studies in a variety of heme systems, we assign the low-frequency coupling in MbCO to torsional motions of the CO molecule. The low-frequency mode coupled to band III (approximately 70 cm-1) is found to lie quite close to the value for the heme-doming motion (approximately 50 cm-1) calculated by using the kinetically determined value of the force constant (17 N/m). Significant inhomogeneous broadening in the Soret region of Mb and Mb* is found to be due to a "nonkinetic" coordinate that we associate with the orientation of the proximal histidine. A "kinetic" coordinate, associated with the equilibrium displacement of the iron atom from the porphyrin plane (a) is found to contribute to the inhomogeneous broadening of both the Soret band and band III. The relaxation of the heme as the system evolves from from Mb* to Mb is followed optically as a function of temperature, and a sharp transition temperature is found at 185 K. The blue shifts of the Soret band and band III as Mb* evolves to Mb are found to be nearly identical (delta v*ABS approximately 140 cm-1) and attributed to changes in the mean value of a between Mb* (a*0) and Mb (a0 = 0.45 A). A simple quadratic model for the coordinate coupling that simultaneously accounts for the observed shift, delta v*ABS, the low-temperature kinetics and the kinetic hole burning predicts a*0 = 0.2 +/- 0.05 A and EA = 16 +/- 2 kJ/mol for the room temperature Arrhenius barrier height at the heme. A simple quantitative method for the analysis of kinetic hole-burning experiments is also developed and applied to recent studies involving quaternary and subunit-specific hemoglobin structures.     

Rates and energetics of tyrosine ring flips in yeast iso-2-cytochrome c

Isotope-edited nuclear magnetic resonance spectroscopy is used to monitor ring flip motion of the five tyrosine side chains in the oxidized and reduced forms of yeast iso-2-cytochrome c. With specifically labeled protein purified from yeast grown on media containing [3,5-13C]tyrosine, isotope-edited one-dimensional proton spectra have been collected over a 5-55 degrees C temperature range. The spectra allow selective observation of the 10 3,5 tyrosine ring proton resonances and, using a two-site exchange model, allow estimation of the temperature dependence of ring flip rates from motion-induced changes in proton line shapes. For the reduced protein, tyrosines II and IV are in fast exchange throughout the temperature range investigated, or lack resolvable differences in static chemical shifts for the 3,5 ring protons. Tyrosines I, III, and V are in slow exchange at low temperatures and in fast exchange at high temperatures. Spectral simulations give flip rates for individual tyrosines in a range of one flip per second at low temperatures to thousands of flips per second at high temperatures. Eyring plots show that two of the tyrosines (I and III) have essentially the same activation parameters: delta H++ = 28 kcal/mol for both I and III; delta S++ = 42 cal/(mol.K) for I, and delta S++ = 41 cal/(mol.K) for III. The remaining tyrosine (V) has a larger enthalpy and entropy of activation: delta H++ - 36 kcal/mol, delta S++ = 72 cal/(mol.K). Tentative sequence-specific assignments for the tyrosines in reduced iso-2 are suggested by comparison to horse cytochrome c.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fe-heme conformations in ferric myoglobin.

X-ray absorption near-edge structure (XANES) spectra of ferric myoglobin from horse heart have been acquired as a function of pH (between 5.3 and 11.3). At pH = 11.3 temperature-dependent spectra (between 20 and 293 K) have been collected as well. Experimental data solve three main conformations of the Fe-heme: the first, at low pH, is related to high-spin aquomet-myoglobin (Mb+OH2). The other two, at pH 11.3, are related to hydroxymet-myoglobin (Mb+OH-), and are in thermal equilibrium, corresponding to high- and low-spin Mb+OH-. The structure of the three Fe-heme conformations has been assigned according to spin-resolved multiple scattering simulations and fitting of the XANES data. The chemical transition between Mb+OH2 and high-spin Mb+OH-, and the spin transition of Mb+OH-, are accompanied by changes of the Fe coordination sphere due to its movement toward the heme plane, coupled to an increase of the axial asymmetry.     

Energetics of the equilibrium between two nucleotide-free myosin subfragment 1 states using fluorine-19 nuclear magnetic resonance

A new fluorine-containing reagent has been synthesized and used to specifically label the reactive sulfhydryl [sulfhydryl-1 (SH1)] of myosin subfragment 1 (S-1). The labeled S-1 (S-1-CF3) demonstrates activated calcium and magnesium adenosinetriphosphatase (ATPase) activities relative to S-1 and a lower potassium ethylenediaminetetraacetate (EDTA) ATPase activity. Maximal effect is obtained with the modification of one thiol per S-1. The 19F NMR spectrum of S-1 CF3 contains only one resonance with a line width of 110 Hz, which implies a rotational correlation time of 2.3 X 10(-7) s. The chemical shift of this resonance is sensitive to temperature, PH, ionic strength, and nucleotides bound in the active site. The temperature dependence of the chemical shift clearly indicates two limiting states for the S-1-CF3 with a highly temperature-dependent equilibrium between 5 and 40 degrees C. The low-temperature state appears to be identical with the state resulting from the binding of Mg.ADP or Mg.AMPPNP at 25 degree C. The energetics of the conformational change have been studied under various conditions. At pH 7 in 25 mM cacodylate, 0.1 M KCl, and 1 mM EDTA, delta H degree = 30 kcal/mol and delta S degree = 105 cal deg-1 mol-1. A decrease in pH to 6.5 results in an increased population of the low-temperature state with delta H degree = 31 kcal/mol and delta S degree = 107 cal deg-1 mol-1. Similarly, the low-temperature state is favored by low ionic strength. In 5.8 mM piperazine-N,N'bis(2-ethanesulfonic acid) and 1 mM EDTA (pH 7), delta H degree = 8 kcal/mol and delta S degree = 27 cal deg-1 mol-1. We have also obtained 19F NMR spectra of S-1-CF3 in D2O solution with 30% ethylene glycol at pH 7.1. Increasing concentrations of ethylene glycol progressively stabilize the high-temperature states.     

Magnetic circular dichroism of myoglobin-thiolate complexes.

Various complexes of myoglobin (Mb) with thiolate were studied by use of magnetic circular dichroism (MCD) spectroscopy. 1. MetMb-ethyl, n-propyl and isopropylmercaptan complexes offered MCD spectra similar to that of cytochrome P-450 (P-450) with respect to shape and intensity ratio of Soret MCD to Q0-0 MCD. The MCD spectra did not show any pH dependence. The complexes reduced by sodium dithionite exhibited the MCD spectrum of deoxyMb, indicative of release of thiolate anion from the heme iron. 2. Cysteine and cysteine methyl ester coordinated to the heme iron at pH 9.18 but not at pH 6.86 and 11.45. The complex formed at pH 9.18 gave an MCD spectrum similar to that of P-450, and an MCD spectrum of deoxy Mb on reduction with sodium dithionite. 3. The 2-mercaptoethanol complex exhibited three A terms associated with the Q0-0-1, and Soret transitions at pH 6.86 similar to those of Fe(II) cytochrome c, which indicates that Mb was reduced by this reagent at pH 6.86. At pH 9.18 2-mercaptoethanol gave an MCD spectrum similar to that of alkyl mercaptan just after the addition. With the time changed into deoxy Mb through some intermediate of reduced Mb-thiolate complex. At pH 11.45 2-mercaptoethanol formed complex which exhibited an MCD spectrum similar to those of other alkylmercaptans. 4. Sodium sulfide gave an MCD spectrum which resembled that of the normal thiol Mb complex just after addition at pH 6.86. The complex was gradually reduced to give 610 nm trough in addition to the MCD of deoxy Mb. The Mb-sulfur complex formed at pH 9.18 was gradually reduced to give an MCD spectrum which was fairly different from that of deoxy Mb. A similar MCD spectrum was observed at pH 11.45 just after the addition of Na2S. These results were considered to suggest the saturation of one of the conjugated double bonds of the porphyrin by sulfur.     

Dynamic equilibrium between the two conformational states of spin-labeled tropomyosin

Tropomyosin was labeled with a maleimide nitroxide spin-label attached to cysteine-190 via a succinimido ring which was subsequently opened by incubation at alkaline pH. Electron spin resonance (ESR) spectra showed a temperature-dependent equilibrium, below the main unfolding transition of tropomyosin, between labels which were restricted in their motion (strongly immobilized), predominating at low temperatures, and those which were highly mobile (weakly immobilized), predominating at higher temperatures. These label states were associated with two protein states from a comparison of the ESR spectral changes with the thermal unfolding profile of tropomyosin. The strongly immobilized labels were associated with the completely folded molded and the weakly immobilized labels with a partially unfolded (in the cysteine-190 region) state which is an intermediate in the thermal unfolding of tropomyosin. A spectral subtraction technique was used to measure the concentration ratio of strongly and weakly immobilized labels from which an equilibrium constant, K, was determined at different temperatures. A linear van't Hoff plot was obtained, indicating that the spin-labeled protein is in thermal equilibrium between these two conformational states with delta H = 17 kcal/mol, delta S = 56 cal/(deg X mol), and K = 1.0 at 34 degrees C. An upper limit of 10(7) s-1 for the conformational fluctuation was estimated from the shapes and separation of the two ESR spectral components. In contrast to the label with the opened succinimido ring, the spin-label with an intact succinimido ring remained strongly immobilized on the protein, indicating that in the partially unfolded state the molecule retains structure in the cysteine-190 region.     

Electron-paramagnetic-resonance studies of leghaemoglobins from soya-bean and cowpea root nodules. Identification of nitrosyl-leghaemoglobin in crude leghaemoglobin preparations

1. Leghaemoglobins from soya-bean (Glycine max) and cowpea (Vigna unguiculata) root nodules were purified by chromatography on DEAE-cellulose phosphate columns at pH8.0 and pH5.8, to avoid the relatively low pH (5.2) commonly used to purify these proteins. 2. E.p.r. (electron-paramagnetic-resonance) spectra of the fluoride, azide, hydroxide and cyanide complexes of these ferric leghaemoglobins were very similar to the spectra of the corresponding myoglobin derivatives, indicating that the immediate environment of the iron in leghaemoglobin and myoglobin is similar, an imidazole moiety of histidine being the proximal ligand to the haem iron [cf. Appleby, Blumberg, Peisach, Wittenberg & Wittenberg (1976) J. Biol. Chem.251, 6090-6096]. 3. E.p.r. spectra of the acid-metleghaemoglobins showed prominent high-spin features very near g=6 and g=2 and, unlike myoglobin, small low-spin absorptions near g=2.26, 2.72 and 3.14. The width of the g=6 absorption derivative at 10-20K was about 4-4.5mT, similar to the value for acid-methaemoglobin. In contrast, a recently published (Appleby et al., 1976) spectrum of acid-metleghaemoglobin a had less high-spin character and a much broader absorption derivative around g=6. 4. E.p.r. spectra of ferric leghaemoglobin nicotinate and imidazole complexes suggest that the low-spin absorption near g=3.14 can be attributed to a trace of ferric leghaemoglobin nicotinate, and those near g=2.26 and 2.72 are from an endogenous dihistidyl haemichrome. 5. A large e.p.r. signal at g=2 in all samples of crude leghaemoglobin was shown to be from nitrosyl-leghaemoglobin. A soya-bean sample contained 27+/-3% of the latter. A previously unidentified form of soya-bean ferrous leghaemoglobin a was shown to be its nitrosyl derivative. If this is not an artifact, and occurs in the root nodule, the nitrosyl radical may interfere with the function of leghaemoglobin.     

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.     

Equilibrium study on the binding between thermolysin and Streptomyces metalloprotease inhibitor, talopeptin (MKI)

The binding between thermolysin and its specific inhibitor, talopeptin (MKI), was found to show a fluorescence increase when excited at 280 nm and 295 nm, and a difference spectrum characterized by two peaks at 294 nm and 285 nm with a shoulder around 278 nm, indicating a microenvironmental change in tryptophan residue(s) of thermolysin and/or talopeptin. The inhibitor constant of talopeptin against thermolysin, Ki, was determined over the pH range 5-9 from the inhibition of the enzyme activity towards 3-(2-furylacryloyl)-glycyl-L-leucine amide (FAGLA) as a substrate. The dissociation constant of thermolysin-talopeptin complex, Kd, determined directly from fluorometric titration was in good agreement with the inhibitor constant, Ki, between pH 6 and 8.5. The pH dependence of Ki and Kd suggested that at least two ionizable groups of thermolysin in their protonated forms are essential for the binding between thermolysin and talopeptin. The temperature dependence of K1 at pH 5.5 indicated that the binding is largely exothermic (delta H degree = -12 kcal/mol) and essentially enthalpy-driven.     

The effect of quaternary structure on the state of the alpha and beta subunits within nitrosyl haemoglobin. Low temperature photodissociation and the ESR spectra.

Photodissociation of nitrosyl haemoglobin and nitrosyl hybrids, in which either the alpha or beta subunit is in the nitrosyl form has been stidued at liquid helium temperature (4.2 degrees K) by electron spin resonance and optical absorption spectroscopy. In the presence of inositol hexaphosphate, the photodissociated form of nitrosyl haemoglobin showed an anomalous absorption spectrum in the near infrared region. The experiments with nitrosyl hybrids showed that the alphaNO subunit within the T state haemoglobin is predominantly responsible for the anomalous photodissociated form and the ESR spectrum with three distinct hypefines. The ESR spectrum of alphaNO2betadeoxy2 with inositol hexaphosphate appeared to be very similar to that of the 5-coordinated NO-haem complexes but the absorption spectrum of its photodissociated form was similar to none of protoporphyrin Fe(II) derivatives so far reported. This result suggests that the anomalous photodissociated form may be attributable to some structural distortion of porphyrin or a new electronic state of the haem with different spin state from that of deoxyhaemoglobin.     

Partitional and motional properties of a spin-labeled daunomycin in lipid bilayers. An ESR study.

The partition coefficient of a spin-labeled daunomycin (DAU-SL) in dimyristoylphosphatidylcholine membrane has been determined using the electron spin resonance (ESR) method. The experiment was carried out as a function of temperature between 5 degrees C and 35 degrees C, giving partition coefficients between 2 and 6 without abrupt change at the phase transition. The thermodynamic parameters on transferring the DAU-SL from the aqueous phase to the lipid bilayer were also calculated. The calculated values are: delta H = 6.11 kcal/mol and delta S = 23 cal/K mol. The partitioning of the DAU-SL and its motion in the membrane were investigated in a wide range of pH (4-10.3). The data show that pH has no effect on partitioning of the DAU-SL which suggest that the drug exists in the uncharged form in the bilayer.     

EPR studies on the photoinduced intermediates of NO complexes in recombinant ferric-Mb trapped at low temperatures

The nitrosyl complex of ferric myoglobin is EPR-silent. Upon photolysis at low temperatures, the photoinduced intermediates trapped in the distal heme cavity exhibit new EPR spectra due to the interaction between the photodissociated NO (S=1/2) and the ferric high spin heme (S=5/2). In order to elucidate the effect of distal E7 (His64) and E11 (Val68) mutations upon the electronic structure of the metal center, its immediate environment, and its interaction with the photodissociated NO, EPR spectra of the photoproducts of the NO complexes of recombinant ferric Mb mutants were measured at 5 K. EPR spectra of the photoproducts were closely related to the size and/or the polarity of the distal pocket residues. The distal pocket of the E7 mutants seemed to be sterically crowded, even decreasing the side chain volume or changing its hydrophobicity by replacing amino acid at position 64. We have found that the mobility of the photodissociated NO molecule in the distal heme pocket was strongly governed by the nature of the amino acid residue at E11 position.     

Electron Spin Resonance Studies of Nitrosyl Haemoglobin in Human Liver, Colon and Stomach Tumour Tissues

Iron nitrosyl haemoglobin (HbFeNO) gives well defined ESR spectra, and can be detected at room temperature, in contrast with most transition metal complexes of biological importance. This is because the unpaired electron remains strongly localised on the NO ligand. It is of importance because it proves the formation of nitric oxide, which unfortunately cannot be detected directly by ESR spectroscopy. We have studied a range of tissues taken from human liver, colon and stomach tumours which have been directly frozen to 77K and studied at 77K. The results show that formation of HbFeNO is rare in tissue adjacent to tumour tissue (“peripheral tissue”), but is always found in necrotic central regions, if present. However, in several cases, HbFeNO was also detected in tumour tissue which was not necrotic. Two factors contribute to the formation of this complex. One is the presence of “free” NO molecules in the cellular regions, and the other is the presence of deoxyferrohaemoglobin, since neither ferrihaemoglobin nor oxyhaemoglobin react to give this complex. [For systems containing myoglobin these comments include the possibility of the formation of nitrosylmyoglobin, which gives very similar ESR spectra.]     

Temperature dependence of Q-band electron paramagnetic resonance spectra of nitrosyl heme proteins

The Q-band (35 GHz) electron paramagnetic resonance (EPR) spectra of nitrosyl hemoglobin (HbNO) and nitrosyl myoglobin (MbNO) were studied as a function of temperature between 19 K and 200 K. The spectra of both heme proteins show two classes of variations as a function of temperature. The first one has previously been associated with the existence of two paramagnetic species, one with rhombic and the other with axial symmetry. The second one manifests itself in changes in the g-factors and linewidths of each species. These changes are correlated with the conformational substates model and associate the variations of g-values with changes in the angle of the N(his)-Fe-N(NO) bond in the rhombic species and with changes in the distance between Fe and N of the proximal (F8) histidine in the axial species.     

Structural dynamics of myoglobin: FTIR-TDS study of NO migration and binding

By using Fourier transform infrared photolysis difference spectroscopy combined with temperature derivative spectroscopy at cryogenic temperatures, we have measured infrared spectra of the stretching absorption on nitric oxide (NO) in the heme-bound and photodissociated states of ferrous and ferric nitrosyl myoglobin (MbNO) and a few site-specific Mb mutants. The NO absorption was utilized as a sensitive local probe of ligand interactions with active-site residues and movements within the protein. By comparison with results obtained in previous spectroscopic and structural studies of carbonmonoxy myoglobin (MbCO), the MbNO data were interpreted in structural terms. In the NO-bound state, conformational heterogeneity was inferred from the appearance of multiple bands, arising from different electrostatic interactions with active site residues, most importantly, His-64. In ferrous MbNO, a primary photoproduct site similar to site B of MbCO was found, as indicated by a characteristic NO stretching spectrum. In ferric MbNO, the His-64 side chain appears to interfere with trapping of NO in this site; only a very weak photoproduct spectrum was observed in Mb variants in which His-64 was present. Upon extended illumination, the photoproduct spectrum changed in a characteristic way, indicating that NO readily migrates to a secondary docking site C, the Xe4 cavity, in which the ligand performs librational motions on the picosecond time scale. This docking site may play a role in the physiological NO scavenging reaction. Surprisingly, NO cannot be trapped at all in secondary docking site D, the Xe1 cavity.     

Electron-spin resonance of nitrosyl haemoglobins: normal alpha and beta chains and mutants Hb M Iwate and Hb Zürich.

At 77 K the electron spin resonance (ESR) spectra of the NO derivatives of the mutant haemoglobins Hb M Iwate and Hb Zurich as well as of the isolated chains of normal haemoglobin were studied. Two types of ESR spectra differing in the g-value and the hyperfine splitting at gzz were observed. The type II spectrum is characterized by a hyperfine structure at gzz = 2.005 with a splitting constant of deltaH = 23 G (14NO) or 32 G (15NO), respectively. In the type I spectrum the splitting constant of the hyperfine structure at gzz = 2.009 amounts to deltaH = 18 G (14NO) or 23 G (15NO), respectively. In some cases this hyperfine structure is coincident with another one at gxx = 2.064 with nearly identical splitting constant. In addition, the type I spectrum is characterized by an increased ESR absorption at gxx = 2.064. At neutral pH the NO derivatives of the isolated chains as well as of the mutant haemoglobins give rise to a type II spectrum. In correspondence with previous results gained with normal NO haemoglobin, the ESR spectra of the NO-alpha chains and NO-Hb Zurich show a transition to type I in the acid region. This transition is favoured by binding of 2,3-bisphosphoglycerate. On the other hand, the ESR spectra of the NO-beta chains and NO-Hb M Iwate are of the type II also at acid pH. The NO-beta chains show a transition of the ESR spectrum from type II to type I only at alkaline pH. These results indicate that in the tetrameric NO haemoglobin only the alpha chains are responsible for the transition of the ESR spectrum from type II to type I in the acid region. The two types of ESR spectra are interpreted in terms of two kinds of haem-NO complexes differing in the iron-NO and iron-imidazole distances. The type II spectrum is attributed to a complex with a relatively short iron-imidazole distance which is responsible for a weakened sigma-bond in trans position. The type I spectrum arises then from a complex with a larger iron-imidazole bond leading to an approach of the NO molecule to the iron. The influence of the protein conformation upon the iron-imidazole bond length is discussed with regard to the ESR spectra of the mutant NO haemoglobins and considering the influence of agents modifying the protein structure.