A steric mechanism for inhibition of CO binding to heme proteins.

The crystal structures of myoglobin in the deoxy- and carbon monoxide-ligated states at a resolution of 1.15 angstroms show that carbon monoxide binding at ambient temperatures requires concerted motions of the heme, the iron, and helices E and F for relief of steric inhibition. These steps constitute the main mechanism by which heme proteins lower the affinity of the heme group for the toxic ligand carbon monoxide.

Differential sympathetic neural control of oxygenation in resting and exercising human skeletal muscle.

Metabolic products of skeletal muscle contraction activate metaboreceptor muscle afferents that reflexively increase sympathetic nerve activity (SNA) targeted to both resting and exercising skeletal muscle. To determine effects of the increased sympathetic vasoconstrictor drive on muscle oxygenation, we measured changes in tissue oxygen stores and mitochondrial cytochrome a,a3 redox state in rhythmically contracting human forearm muscles with near infrared spectroscopy while simultaneously measuring muscle SNA with microelectrodes. The major new finding is that the ability of reflex-sympathetic activation to decrease muscle oxygenation is abolished when the muscle is exercised at an intensity > 10% of maximal voluntary contraction (MVC). During high intensity handgrip, (45% MVC), contraction-induced decreases in muscle oxygenation remained stable despite progressive metaboreceptor-mediated reflex increases in SNA. During mild to moderate handgrips (20-33% MVC) that do not evoke reflex-sympathetic activation, experimentally induced increases in muscle SNA had no effect on oxygenation in exercising muscles but produced robust decreases in oxygenation in resting muscles. The latter decreases were evident even during maximal metabolic vasodilation accompanying reactive hyperemia. We conclude that in humans sympathetic neural control of skeletal muscle oxygenation is sensitive to modulation by metabolic events in the contracting muscles. These events are different from those involved in either metaboreceptor muscle afferent activation or reactive hyperemia.

Intensity of highly anisotropic low-spin heme EPR signals.

A semi-empirical formula has been derived to calculate the concentration of low-spin heme compounds that are highly anisotropic, i.e. 3 less than gz less than 4, and where information only on the gz absorption is available.

A proton nuclear magnetic resonance study of sulfmyoglobin cyanide.

The proton nuclear magnetic resonance spectrum of sulfmyoglobin cyanide was studied at 400 MHz. The position of a methyl-group resonance at low field is consistent with a chlorin-like structure for the prosthetic group. The proton NMR spectrum of the cyanide derivative of the purified prosthetic group which decomposes upon extraction from the protein was found to be the same as that of the cyanide derivative of the prosthetic group extracted from myoglobin and a sample prepared from hemin-Cl.

Adiabatic compressibility of myoglobin. Effect of axial ligand and denaturation.

An ultrasonic technique has been employed to study the adiabatic compressibility of three metmyoglobin derivatives (aquomet-, fluoromet- and azidometmyoglobin) at neutral pH, and aquometmyoglobin as a function of pH in the frequency range of 1-10 MHz at 20 degrees C. No difference was observed in the adiabatic compressibility of the various derivatives. This indicates that the binding of different axial ligands to myoglobin does not affect significantly the conformational fluctuations of the protein. The finding is consistent with the results of the hydrogen exchange rate experiment, indicating that both types of measurements are useful for the study of protein dynamics. Upon acid-induced denaturation, the adiabatic compressibility of myoglobin drops from 5.3 X 10(-12) cm2/dyn to 0.5 X 10(-12) cm2/dyn. Plausible reasons for such a decrease are discussed.

High magnetic field Mössbauer studies of deoxymyoglobin, deoxyhemoglobin, and synthetic analogues.

Mössbauer spectra of deoxymyoglobin, deoxyhemoglobin, and the synthetic analogues, iron (II) 2-methylimidazole meso-tetraphenylporphyrin, and iron (II) 1,2-dimethylimidazole meso-tetraphenylporphyrin have been observed in high magnetic fields and over a wide range of temperature. At temperatures greater than 20 K all materials exhibit remarkably similar spectra, with anisotropic internal magnetic fields decreasing as 1/T. All have negative quadrupole interaction, and both this and the magnetic anisotropy imply that the orbital of the odd electron is prolate in the ground quintet, with little unquenched orbital angular momentum. At 4.2 K the spectra differ, suggesting different detailed structure within the quintet. In contrast to the proteins, the 2-methyl model exhibits spectra at 4.2 K which imply that the lowest spin state has high susceptibility in a single direction.

13C-NMR study of labeled vinyl groups in paramagnetic myoglobin derivatives.

The 13C-NMR spectra of high-spin met-aquo myoglobin, spin-equilibrium met-azido myoglobin, low-spin met-cyano myoglobin, deoxy myoglobin and carbonmonoxy myoglobin from sperm whale reconstituted with hemin 13C enriched at both vinyl alpha or beta positions have been recorded. In all cases the labeled vinyl 13C signals are clearly resolved and useful spectra could be obtained within approx. 15 minutes. The decoupling of multiplet structure due to attached proton(s) has led to the specific assignment of vinyl 13C alpha signals in all paramagnetic derivatives and the 13C beta signals in met-cyano myoglobin. In all other cases, the collapse of the proton multiplet structure as a function of 1H decoupling frequency has located, but not assigned, the attached 1H resonance positions which are obscured by the intense diamagnetic envelope in the 1H-NMR spectrum. The resulting vinyl 13C hyperfine shifts follow Curie behavior, and the patterns closely resemble those in the appropriate model complexes in the same oxidation/spin/ligation state, except that the protein exhibits more in-plane asymmetry. The hyperfine shift patterns are indicative of dominant pi contact shifts for all ferric complexes. Deoxy myoglobin vinyl 13C and 1H contact shifts provide little evidence for pi bonding.

Infrared magnetic circular dichroism of myoglobin derivatives.

By use of a newly constructed CD instrument, infrared magnetic circular dichroism (MCD) spectra were observed for various myoglobin derivatives. The ferric high spin myoglobin derivatives such as fluoride, water and hydroxide complexes, commonly exhibited the MCD spectra consisting of positive A terms. Therefore, the results reinforced the assignment that the infrared band is the charge transfer transition to the degenerate excited state (eg (dpi)). Since the fraction of A term estimated was approximately 80% for myoglobin fluoride and approximately 35% for myoglobin water, the effective symmetry for myoglobin fluoride is determined to be as close as D4h, while that for myoglobin water seems to have lower symmetry components. The ferric low spin derivatives such as myoglobin cyanide, myoglobin imidazole and myoglobin azide showed positive MCD spectra which are very similar to the electronic absorption spectra. These MCD spectra were assigned to the charge transfer transitions from porphyrin pi to iron d orbitals on the ground that they were observed only for the ferric low spin groups and insensitive to the axial ligands. The lack of temperature dependence in the MCD magnitude indicated that the MCD spectra are attributable to the Faraday B terms. Deoxymyoglobin, the ferrous high spin derivative, had fairly strong positive MCD around 760 nm with an anisotropy factor (delta epsilon/epsilon) of 1.4-10(-4). It shows some small MCD bands from 800 to 1800 nm. Among the ferrous low spin derivatives, carbonmonoxymyoglobin did not give any observable MCD in the infrared region while oxymyoglobin seemed to have significant MCD in the range from 700 to 1000 nm.

EPR studies on the photoproducts of manganese(II) protoporphyrin-IX substituted myoglobin nitrosyl complexes trapped at low temperature: effects of site-specific chemical modification of the distal histidine on ligand-binding structures.

Manganese(II) protoporphyrin-IX substituted myoglobin with site-specifically cyanated or N-tetrazolated distal histidine (His) was prepared and low-temperature photolysis of nitric oxide (NO) from their nitrosyl complexes was examined by electron paramagnetic resonance (EPR) spectroscopy in order to elucidate the steric crowding of the distal heme moiety. The photoproduct of NO complex of the tetrazolated Mn(II)Mb (tetrazole-Mn(II)Mb) exhibited widespread absorption in the magnetic field from zero to 0.4 T due to a spin-coupled interaction between the high-spin Mn(II) center (S = 5/2) and the photodissociated NO (S = 1/2) trapped adjacent to the metal center. This indicates that the NO complex of tetrazole-Mn(II)Mb has sterically restricted distal heme pocket. On the other hand, the photoproduct of NO complex of cyanated Mn(II)Mb (BrCN-Mn(II)Mb) exhibited only the broad g = 6 absorption due to the magnetic dipole-dipole interaction between the photodissociated NO and the high-spin Mn(II) center. A drastic conformational change in the heme-ligand moiety, in which the distal histidine side chain is pushed toward the outside of the heme pocket, leaving an open space in the distal heme pocket, can be suggested.

NMR study of the active site of shark met-cyano myoglobins.

The myoglobins from the sharks Galeorhinus japonicus and Musterus japonicus possess a distal Gln-E7 instead of the usually found His-E7. The met-cyano form of these shark myoglobins has been studied by 1H- and 15N-NMR in order to gain insight into the functional properties of the Gln-E7. The analysis of paramagnetic relaxation has provided the assignment of the resonance arising from one of the Gln-E7 N epsilon H labile protons, whilst the rate of its chemical exchange has been analyzed in detail by using a saturation transfer method. The hydrogen-bonding interaction between this proton and Fe-bound-CN(-) has been clearly manifested in the hyperfine shift of the Gln-E7 N epsilon H proton resonance as well as its chemical exchange behavior. The resonances of the Gln-E7 side-chain non-labile protons have been partly assigned on a basis of both scalar and dipolar connectivities. The analysis of the dipolar connectivities among the side-chain protons and the iron-proton distances determined from their paramagnetic relaxation rate has revealed that the side chain adopts a conformation with its carbonyl oxygen oriented away from the heme. Although (1)H-NMR spectra of these two myoglobins are essentially similar, a relatively large difference in the shift of Gln-E7 N(epsilon)H proton and Fe-bound C15 N- resonances between the two has been observed which is attributed to a differential hydrogen-bonding interaction between these proteins. The present study demonstrates the sensitivity of NMR parameters to the hydrogen-bonding interaction between coordinated ligand and a distal amino-acid side chain in paramagnetic hemoproteins.

A chemometric analysis of the resonance Raman spectra of mutant carbonmonoxy-myoglobins reveals the effects of polarity.

Resonance Raman spectra of 10 carbonmonoxy-myoglobins have been obtained, including sperm whale native, pig wild-type, and the mutants H64L, H64A, V68T, V68N, H64V/V68T, F43W, F46V, and L29F. This series was chosen in order to study the effect of ligand binding pocket polarity on the positions of the v(Fe-CO) and delta (Fe-C-O) bands. Spectra of both 12CO and 13CO isotopic forms have been obtained and a detailed analysis has facilitated the identification of both the ligand-specific bands and six underlying porphyrin bands which are insensitive to this isotopic substitution. Along with a band-fitting analysis of infrared spectra, these resonance Raman data provide a comprehensive evaluation of the vibrations of the FeCO unit. The band positions of the ligand-specific modes are found to depend on the structure of the ligand binding pocket, arising from the strength of back-bonding within the FeCO unit, and clear correlations exist between the v(Fe-CO), delta (Fe-C-O), and v(C-O) band positions which characterize this synergic bonding. The results are consistent with the proposal that the vibration band positions are determined primarily by the electrostatic potential at the ligand. Five discrete band sets are observed for this set of mutants, suggesting that 5 discrete conformations occur.

Myoglobin signal as an NMR tissue thermometer: implication for hyperthermia treatment of tumors.

Measuring local tissue temperature is critical in establishing a rational approach for hyperthermia treatment of tumors. We have found that the heme signals of myoglobin provide a unique basis for NMR thermometry in vivo. In particular the 5-methyl heme signal of MbCN exhibits a sharp, temperature-dependent resonance that is distinguishable in the tissue spectrum. Its calibrated chemical shift can then reflect the local tissue temperature in vivo.

The energy level scheme for the ferryl heme in compound II of the peroxidase-catalase family as determined from analysis of low-temperature magnetic circular dichroism.

The expressions for temperature-dependent magnetic circular dichroism (MCD) of the ferryl heme (Fe(4+)Por, S=1), which is a model of an intermediate product of the catalytic cycle of heme enzymes (compound II), have been derived in the framework of a two-term model. Theoretical predictions for the temperature and magnetic field dependence of MCD intensity of the ferryl heme are compared with those of the high-spin and low-spin ferric heme. Analysis of reported MCD spectra of myoglobin peroxide [Foot et al., Biochem. J. 2651 (1989) 515-522] and compound II of horseradish peroxidase [Browett et al., J. Am. Chem. Soc. 110 (1987) 3633-3640] has shown the presence in the samples of approximately 1% of a low-spin ferric component, which, however, should be taken into account in simulating observed temperature dependences of MCD intensity. The values of two adjustable parameters are estimated from the fit of the observed and simulated plots of MCD intensity against the reciprocal of the absolute temperature. One of them, the energy gap between the ground and excited terms, predetermines the axial zero-field splitting. The other parameter is correlated with the energy of splitting of excited quartets arising from either the porphyrin pi-->pi* transition or the spin-allowed charge-transfer transition.

Solvent isotope effects on NMR spectral parameters in high-spin ferric hemoproteins: an indirect probe for distal hydrogen bonding.

The influence of solvent isotope composition on 1H-NMR resonance position and linewidth of heme methyls has been investigated for a variety of high-spin ferric hemoproteins for the purpose of detecting hydrogen-bonding interactions in the heme cavity. Consistently larger hyperfine shifts and paramagnetic linewidths in 2H2O than 1H2O are observed for metmyoglobins and methemoglobin possessing a coordinated water molecule. The analysis of the dynamics of labile proton exchange in sperm whale metmyoglobin, and the absence of any isotope effects in the five-coordinate Aplysia metmyoglobin, indicate that the significant axial modulation of heme electronic structure by solvent isotope is consistent with arising from distal hydrogen-bonding interactions. The presence or absence of similarly large isotope effects on shifts and linewidths in other hemoproteins, depending on the presence of a bound water in the distal heme pocket, suggests that this isotope effect can serve as a probe for the presence of such bound water. The absence of any detectable isotope effect on either shifts or linewidths in resting-state horseradish peroxidase supports a five-coordinate structure with bound water absent from the vicinity of the iron.

Effect of nitrosylmyoglobin and saturated fatty acid anions on metmyoglobin-catalyzed oxidation of aqueous methyl linoleate emulsions.

In aqueous methyl linoleate emulsions (pH 7.4, 25 degrees C, air-saturated), nitrosylmyoglobin and saturated fatty acid anions (palmitate and stearate investigated) each showed antioxidant effect on metmyoglobin-induced peroxidation as measured by oxygen depletion rate. For equimolar concentration of nitrosylmyoglobin and metmyoglobin and for metmyoglobin in moderate excess, a reduction in oxygen consumption rate of approximately 70% was observed. Fatty acid anions reduced oxygen consumption rate most significantly for palmitate (up to 60% for a fatty acid:heme protein ratio of 90:1). No further antioxidative effect was seen for fatty acid anions in the presence of nitrosylmyoglobin, whereas nitrosylmyoglobin showed a further antioxidant effect in presence of fatty acid anions in the metmyoglobin-catalyzed process. The antioxidative mechanism of nitrosylmyoglobin and fatty acid anions is different, and while the fatty acid anions seem active in inhibiting initiation of oxidation through protection against metmyoglobin activation into perferrylmyoglobin, as shown by freeze-quench Electron Spin Resonance (ESR) spectroscopy, nitrosylmyoglobin is rather active in the oxygen consuming (propagation) phase.

Conversion of metmyoglobin to NO myoglobin in the presence of nitrite and reductants.

Formation of NO myoglobin through the reaction of horse heart metmyoglobin with NADH in the presence of nitrite was observed optically at pH 5.5. Superoxide generation during the reaction was demonstrated using the ESR spin trap, 5,5-dimethyl-1-pyrroline-1-oxide. A weak optical spectrum corresponding to oxymyoglobin appeared transiently and the spectrum of NO myoglobin then developed. The conversion to NO myoglobin was eliminated in the presence of catalase, SOD or 5,5-dimethyl-1-pyrroline-1-oxide. The kinetics of NADH oxidation and oxygen consumption catalyzed by myoglobin showed an initial lag phase, indicating a chain reaction. When the oxygen was exhausted, the NO form emerged. The duration of the lag phase was prolonged by an increase in the concentration of catalase, SOD or 5,5-dimethyl-1-pyrroline-1-oxide, whereas it disappeared in the presence of H2O2. The spectral change from metmyoglobin to NO myoglobin was also observed under anaerobic conditions though the rate was slower than that obtained under aerobic conditions, while the spectral change was accelerated in the presence of H2O2. Nitric oxide (NO) was derived through the reaction of nitrite with NADH. The formation of NO myoglobin from metmyoglobin is explained in terms of the NADH-oxidase reaction catalyzed by myoglobin. Ascorbate and GSH also serve as reductants though NO myoglobin was formed slowly.

X-ray crystal structure of the fluoride derivative of Aplysia limacina ferric myoglobin at 2.0 A resolution. Stabilization of the fluoride ion by hydrogen bonding to Arg66 (E10).

The X-ray crystal structure of the fluoride derivative of Aplysia limacina ferric myoglobin has been solved and refined at 2.0 A resolution; the crystallographic R-factor is 13.6%. The fluoride ion binds to the sixth co-ordination position of the heme iron, 2.2 A from the metal. Binding of the negatively charged ligand on the distal side of the heme pocket of this myoglobin, which lacks the distal His, is associated with a network of hydrogen bonds that includes the fluoride ion, the residue Arg66 (E10), the heme propionate III, three ordered water molecules and backbone or side-chain atoms from the CD region. A comparison of fluoride and oxygen dissociation rate constants of A. limacina myoglobin, sperm whale (Physeter catodon) myoglobin and Glycera dibranchiata monomeric hemoglobin, suggests that the conformational readjustment of Arg66 (E10) in A. limacina myoglobin may represent the molecular basis for ligand stabilization, in the absence of a hydrogen-bond donor residue at the distal E7 position.

Deoxymyoglobin studied by the conformational normal mode analysis. II. The conformational change upon oxygenation.

The conformational change taking place in myoglobin concomitantly with the observed geometrical change at the heme-His(F8) linkage upon oxygenation is studied by normal mode analysis, which is based on the quadratic approximation of the conformational energy function. The heme-globin interaction energy increases for this change by 8.114 kcal/mol when both the heme group and the globin molecule are held rigid. When they are permitted flexibility, the interaction energy relaxes by 7.038 kcal/mol, and the difference (1.076 kcal/mol) is distributed as strain energy within the molecule. This increase is the work necessary for the heme group to move against the force exerted by the globin. If the force is assumed to be invariable during this move, the work is small, 0.276 kcal/mol, meaning that the force is strongly variable. Furthermore, this means that the heme group is located near the equilibrium point of the potential energy of the heme-globin interaction. The change in the localized strain energy stored in the force field at the linkage between the heme and the imidazole of HisF8 is estimated to be of the same order of magnitude as the distributed energy. The largest atomic displacements are observed at the region from the F helix to the beginning of the G helix, and secondary large displacements occur at several regions, i.e, the A helix, from the C helix to the CD corner, the E helix, and the C-terminal side of the H helix. All of these regions have strong dynamic interactions with the heme group, either directly or indirectly. Their secondary structures show complex deformations. In other parts, relatively rigid segments undergo rotational and/or bending changes in a way consistent with the large changes described above and close atomic packing within the molecule. The calculated conformational change is decomposed to vibrational normal modes of deoxymyoglobin. The magnitude of the conformational change, measured by the mass-weighted mean-square atomic displacement, is accounted for up to 92.0% by the 151 normal modes with frequencies lower than 40 cm-1. In descending order of contribution, the first six modes, each of which has a frequency lower than 12 cm-1, account for up to 57.4%. This means that the functionally important conformational change can well be expressed in terms of a relatively small number of collective low frequency normal modes.

Structure of myoglobin-ethyl isocyanide. Histidine as a swinging door for ligand entry.

The structure of myoglobin(Fe II)-ethyl isocyanide has been solved at 1.68 A resolution by X-ray crystallography. The isocyano group of the ligand is distorted from the linear conformation observed in solution and in model compounds. Local changes in the protein conformation are also seen. The side-chain of Arg-CD3 moves out into the solvent, and the side-chain of His-E7 swings up and away from the ligand. Both of these side-chains show disorder indicative of dynamic behavior. These outward movements of His-E7 and Arg-CD3 side-chains clear a path from the solvent to the heme iron, suggesting a mechanism for ligand entry.

Deoxymyoglobin studied by the conformational normal mode analysis. I. Dynamics of globin and the heme-globin interaction.

Dynamic properties of deoxymyoglobin are studied theoretically by the analysis of conformational fluctuations. Root-mean-square atomic fluctuations and distance fluctuations between different segments reveal the mechanical construction of the molecule. Eight alpha-helices behave as relatively rigid bodies and corner regions are more flexible, showing larger fluctuations. More particularly, corner regions EF and GH are specific in that flanking alpha-helices extend their rigidity up to a point in the corner region and the two rigid segments are connected flexibly at that point. The FG corner is exceptional. A segment from the F helix to the beginning of the G helix, in which the FG corner is included, becomes relatively rigid by means of strong interactions with the heme group. The whole myoglobin molecule is divided into two large units of motion, one extending from the B to the E helix, and the other from the F to the H helix. These two units are connected covalently by the EF corner. However, dynamic interactions between these two units take place mainly through contacts between helices B and G and not through the EF corner. From correlation coefficients between fluctuational motions of residues and the heme group, 55 residues are identified as having strong dynamic interactions with the heme moiety. Among them, 18 residues in the three segments, one consisting of residues from the C helix to the CD corner, a second consisting of the E helix, and a third from the F helix to the beginning of the G helix, are in close contact with the heme group. Twenty-two of the 55 residues are within four residues of the 18 residues in their sequential residue number and are more than 3 A away from the heme group. The other 15 residues are located further in the sequential residue number and are all found in helices A and H. They are more than 6 A away from the heme group. By the use of correlation coefficients of fluctuations between residues, it is found that dynamic interaction with the heme group is transmitted to the A helix and the beginning of the H helix in the direction Leu(E15)----[Val(All) and Trp(A12)]. The transmission to the C-terminal end of the H helix is mediated by a long segment, from the end of the EF corner to the beginning of the G helix, that lies on the heme group and has close contacts over a wide range.(ABSTRACT TRUNCATED AT 400 WORDS)