Spectroscopic and electrochemical studies of horse myoglobin in dimethyl sulfoxide

This paper reports the first report of rapid, reversible direct electron transfer between a redox protein, specifically, horse myoglobin, and a solid electrode substrate in nonaqueous media and the spectroscopic (UV-vis, fluorescence, and resonance Raman) characterization of the relevant redox forms of myoglobin (Mb) in dimethyl sulfoxide (DMSO). In DMSO, the heme active site of metmyoglobin (metMb) appears to remain six-coordinate high-spin, binding water weakly. Changes in the UV-fluorescence spectra for metMb in DMSO indicate that the protein secondary structure has been perturbed and suggest that helix A has moved away from the heme. UV-vis and RR spectra for deoxyMb in DMSO suggest that the heme iron is six-coordinate low-spin, most likely coordinating DMSO. Addition of CO to deoxyMb in DMSO produces a single, photostable six-coordinate CO adduct. UV-vis and RR for Mb-CO in DMSO are consistent with a six-coordinate low-spin heme iron binding His93 weakly, if at all. The polarity of the distal heme pocket is comparable to that of the closed form of horse Mb-CO in aqueous solution, pH 7. Direct electron transfer between horse Mb and Au in DMSO solution was investigated by cyclic voltammetry. Mb exhibits stable and well-defined electrochemical responses that do not appear to be affected by the water content (1.3-7.5%). The electrochemical characteristics are consistent with a one-electron, quasi-reversible, diffusion-controlled charge transfer process at Au. E degrees for horse Mb in DMSO at Au is -0.241+/-0.005 V vs. NHE. The formal heterogeneous electron transfer rate constant, calculated from delta E(p) at 20 mV/s, is 1.7+/-0.5 x 10(-4) cm/s. The rate, which is unaffected by the presence of 1.3-7.5% water, is competitive with that previously reported for horse Mb in aqueous solution.     

1H-NMR comparative study of the active site in shark (Galeorhinus japonicus), horse, and sperm whale deoxy myoglobins.

1H-NMR spectra of deoxy myoglobins (Mbs) from shark (Galeorhinus japonicus), horse, and sperm whale have been studied to gain insights into their active site structure. It has been demonstrated for the first time that nuclear Overhauser effect (NOE) can be observed between heme peripheral side-chain proton resonances of these paramagnetic complexes. Val-E11 methyl and His-F8 C delta H proton resonances of these Mbs were also assigned from the characteristic shift and line width. The hyperfine shift of the former resonance was used to calculate the magnetic anisotropy of the protein. The shift analysis of the latter resonance, together with the previously assigned His-F8 N delta H proton resonance, revealed that the strain on the Fe-N epsilon bond is in the order horse Mb approximately whale Mb < shark Mb and that the hydrogen bond strength of the His-F8 N delta H proton to the main-chain carbonyl oxygen in the preceding turn of the F helix is in the order shark Mb < horse Mb < whale Mb. Weaker Feporphyrin interaction in shark Mb was manifested in a smaller shift of the heme methyl proton resonance and appears to result from distortion of the coordination geometry in this Mb. Larger strain on the Fe-N epsilon bond in shark Mb should be to some extent attributed to its lowered O2 affinity (P50 = 1.1 mmHg at 20 degrees C), compared to whale and horse Mbs.     

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.     

Kinetics of dithionite reduction of the heme nonapeptide of cytochrome c

The kinetics of dithionite reduction of the oxidized heme nonapeptide fragment of horse heart cytochrome c have been measured as a function of ionic strength at pH 7 and pH 9 by the stopped-flow technique. Dithionite concentration dependences indicate that the radical anion monomer, SO2-., is the active reductant. The pH 7 ionic strength dependence suggests that the heme peptide is reacting as a negatively charged molecule (its overall charge is calculated to be -1). Comparison of these results with the known rate of dithionite reduction of cytochrome c indicates that the heme nonapeptide has substantially greater inherent reactivity than cytochrome c, perhaps due to the greater accessibility of the heme.     

Transient spectroscopy of the reaction of cyanide with ferrous myoglobin. Effect of distal side residues

The reaction of cyanide metmyoglobin with dithionite conforms to a two-step sequential mechanism with formation of an unstable intermediate, identified as cyanide bound ferrous myoglobin. This reaction was investigated by stopped-flow time resolved spectroscopy using different myoglobins, i.e. those from horse heart, Aplysia limacina buccal muscle, and three recombinant derivatives of sperm whale skeletal muscle myoglobin (Mb) (the wild type and two mutants). The myoglobins from horse and sperm whale (wild type) have in the distal position (E7) a histidyl residue, which is missing in A. limacina Mb as well as the two sperm whale mutants (E7 His----Gly and E7 His----Val). All these proteins in the reduced form display an extremely low affinity for cyanide at pH less than 10. The differences in spectroscopy and kinetics of the ferrous cyanide complex of these myoglobins indicate a role of the distal pocket on the properties of the complex. The two mutants of sperm whale Mb are characterized by a rate constant for the decay of the unstable intermediate much faster than that of the wild type, at all pH values explored. Therefore, we envisage a specific role of the distal His (E7) in controlling the rate of cyanide dissociation and also find that this effect depends on the protonation of a single ionizable group, with pK = 7.2, attributed to the E7 imidazole ring. The results on A. limacina Mb, which displays the slowest rate of cyanide dissociation, suggests that a considerable stabilizing effect can be exerted by Arg E10 which, according to Bolognesi et al. (Bolognesi, M., Coda, A., Frigerio, F., Gatti, C., Ascenzi, P., and Brunori, M. (1990) J. Mol. Biol. 213, 621-625), interacts inside the pocket with fluoride bound to the ferric heme iron. A mechanism of control for the rate of dissociation of cyanide from ferrous myoglobin, involving protonation of the bound anion, is discussed.     

Acid Bohr effects in myoglobin characterized by proton NMR hyperfine shifts and oxygen binding studies.

Proton NMR studies of sperm whale and horse deoxymyoglobin have revealed that both proteins exhibit a single, well defined, pH-induced structural change. The changes in hyperfine shifts are clearly observed not only at the heme peripheral substituents, but also at the proximal histidyl imidazole, which suggest that heme-apoprotein contacts are looser in the acidic than alkaline conformations. The hyperfine shift changes are modulated by a single titratable group with a pK of approx. 5.7 in both proteins. Oxygen binding studies of sperm whale myoglobin over a range of temperature and pH showed that, while the oxygen affinity was independent of pH at 25 degrees C, it increased below pH 7 at 0 degrees C and decreased below pH 7 at 37 degrees C. Hence, sperm whale myoglobin exhibits a small acid Bohr effect which most likely arises from the characterized structural changes in the deoxy proteins. While horse myoglobin failed to exhibit a resolvable acid Bohr effect between 0 and 37 degrees C, it did show a weak alkaline Bohr effect at 25 degrees C which disappeared at lower temperatures. Since the oxygen affinity changed smoothly over several pH units, this alkaline Bohr effect can not be associated with any well defined conformational change detected by NMR.     

Induction of redox instability of bovine myoglobin by adduction with 4-hydroxy-2-nonenal

The redox stability of myoglobin (Mb) is compromised by many factors, including lipid oxidation and its products. 4-Hydroxy-2-nonenal (HNE) is an alpha,beta-unsaturated aldehyde derived from the oxidation of omega-6 polyunsaturated fatty acids and is highly reactive and cytotoxic. Our objective was to study potential binding of HNE to Mb and determine how it affects redox stability. OxyMb (0.15 mM) was incubated with HNE (1 mM) at 4, 25, and 37 degrees C at pH 7.4 or 5.6. Samples were analyzed for MetMb formation and by Western blot analyses, LC-MS, LC-MS-MS, circular dichroism (CD), and differential scanning calorimetry (DSC). MetMb formation increased with increasing temperature and was greater at pH 5.6 than at pH 7.4 (P < 0.05). At 37 degrees C, HNE accelerated oxidation at pH 7.4 but not at pH 5.6 (P < 0.05). At both 25 and 4 degrees C, HNE accelerated oxidation at pH 7.4 and 5.6 (P < 0.05). LC-MS revealed the covalent binding of HNE to Mb at both pH values via Michael addition, while Western blot analysis indicated that HNE was bound to histidine (HIS) residues. LC-MS-MS identified six histidine residues of Mb that were readily adducted by HNE, including the proximal (HIS 93) and distal (HIS 64) histidine associated with the heme group. Secondary structure differences between control Mb and Mb incubated with HNE were not detected by CD. However, DSC revealed a decreased T(m) for Mb reacted with HNE at pH 7.4, indicating Mb tertiary structure was altered in a manner consistent with destabilization. These results suggest that HNE accelerates bovine skeletal muscle OxyMb oxidation in vitro by covalent modification at histidine residues.     

Heme protein fluorescence versus pressure.

Fluorescence spectra of several ferric heme proteins have been measured vs. pressure to 6,000 bars. Sperm whale myoglobin (SW Mb), Aplysia myoglobin, leghemoglobin (Lb), and cytochrome P450 all show excitation and emission spectra characteristic of tryptophan in proteins with peak emission at 330-340 nm. At one bar, the fluorescence is weak due to energy transfer to the heme group, which makes the yield a sensitive probe of protein unfolding at high pressure. After an initial decrease of a few percent per kbar, the protein shows a large increase in fluorescence at high pressure. The increase is pH dependent and the results indicate that several high pressure states occur. For SW Mb at 15 degrees C an increase of a factor of 20 occurs with midpoint at 2,000 bars at pH 5 and is only partially reversible, while the increase at pH 7 occurs at 4,000 bars and is only half as large and is completely reversible. Aplysia Mb and Lb show a similar effect, but unfold at a higher pressure than SW Mb. P450 also shows a transition to a state of higher fluorescence, but the transition in this case is irreversible as a stable form, P420, is formed. The fluorescence intensity measurements permit an estimation of the increase in the TRY-heme distance in the high pressure state.     

Redox chemistry and acid-base equilibria of mitochondrial plant cytochromes c

Mitochondrial cytochromes c from spinach, cucumber, and sweet potato have been investigated through direct electrochemical measurements and electronic and 1H NMR spectroscopies, under conditions of varying temperature and pH. The solution behaviors of these plant cytochromes closely resemble, but do not fully reproduce, those of homologous eukaryotic species. The reduction potentials (E0') at pH 7 and 25 degrees C are +0.268 V (spinach), +0.271 V (cucumber), and +0.274 V (sweet potato) vs SHE. Three acid-base equilibria have been determined for the oxidized proteins with apparent pKa values of 2.5, 4.8, and 8.3-8.9, which are related to disruption of axial heme ligation, deprotonation of the solvent-exposed heme propionate-7 and replacement of the methionine axially bound to the heme iron with a stronger ligand, respectively. The most significant peculiarities with respect to the mammalian analogues include: (i) less negative reduction enthalpies and entropies (Delta S0'rc and Delta H0'rc) for the various protein conformers [low- and high-T native (N1 and N2) and alkaline (A)], whose effects at pH 7 and 25 degrees C largely compensate to produce E degrees ' values very similar to those of the mammalian proteins; (ii) the N1 --> N2 transition that occurs at a lower temperature (e.g., 30-35 degrees C vs 50 degrees C at pH 7. 5) and at a lower pH (7 vs 7.5); and (iii) a more pronounced temperature-induced decrease in the pKa for the alkaline transition which allows observation of the alkaline conformer(s) at pH values as low as 7 upon increasing the temperature above 40 degrees C. Regarding the pH and the temperature ranges of existence of the various protein conformers, these plant cytochromes c are closer to bacterial cytochromes c2.     

Effect of pH on axial ligand coordination of cytochrome c" from Methylophilus methylotrophus and horse heart cytochrome c

The effect of protons on the axial ligand coordination and on structural aspects of the protein moiety of cytochrome c' ' from Methylophilus methylotrophus, an obligate methylotroph, has been investigated down to very low pH (i.e., 0.3). The unusual resistance of this cytochrome to very low pH values has been exploited to carry out this study in comparison with horse heart cytochrome c. The experiments were undertaken at a constant phosphate concentration to minimize the variation of ionic strength with pH. The pH-linked effects have been monitored at 23 degrees C in the oxidized forms of both cytochromes by following the variations in the electronic absorption, circular dichroism and resonance Raman spectra. This approach has enabled the conformational changes of the heme surroundings to be monitored and compared with the concomitant overall structural rearrangements of the molecule. The results indicate that horse heart cytochrome c undergoes a first conformational change at around pH 2.0. This event is possibly related to the cleavage of the Fe-Met80 bond and a likely coordination of a H(2)O molecule as a sixth axial ligand. Conversely, in cytochrome c" from M. methylotrophus, a variation of the axial ligand coordination occurs at a pH that is about 1 unit lower. Further, it appears that a concerted cleavage of both His ligands takes place, suggesting indeed that the different axial ligands present in horse heart cytochrome c (Met/His) and in cytochrome c" from M. methylotrophus (His/His) affect the heme conformational changes.     

Structural determinants of fluoride and formate binding to hemoglobin and myoglobin: crystallographic and 1H-NMR relaxometric study.

The x-ray crystal structure of the fluoride derivative of ferric sperm whale (Physeter catodon) myoglobin (Mb) has been determined at 2.5 A resolution (R = 0.187) by difference Fourier techniques. The fluoride anion, sitting in the central part of the heme distal site and coordinated to the heme iron, is hydrogen bonded to the distal His(64)E7 NE2 atom and to the W195 solvent water molecule. This water molecule also significantly interacts with the same HisE7 residue, which stabilizes the coordinated fluoride ion. Moreover, fluoride and formate binding to ferric Aplysia limacina Mb, sperm whale (Physeter catodon) Mb, horse (Caballus caballus) Mb, loggerhead sea turtle (Caretta caretta) Mb, and human hemoglobin has been investigated by 1H-NMR relaxometry. A strong solvent proton relaxation enhancement is observed for the fluoride derivatives of hemoproteins containing HisE7. Conversely, only a small outer-sphere contribution to the solvent relaxation rate has been observed for all of the formate derivatives considered and for the A. limacina Mb:fluoride derivative, where HisE7 is replaced by Val.     

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.     

Two extracellular proteins with alkaline peroxidase activity, a novel cytochrome c and a catalase-peroxidase, from Bacillus sp. No.13

A novel cytochrome c and a catalase-peroxidase with alkaline peroxidase activity were purified from the culture supernatant of Bacillus sp. No.13 and characterized. The cytochrome c exhibited absorption maxima at 408 nm (Soret band) in its oxidized state, and 550 (alpha-band), 521 (beta-band), and 415 (Soret band) nm in its reduced state. The native cytochrome c with a relative molecular mass of 15,000 was composed of two identical subunits. The cytochrome c showed over 50 times higher peroxidase activity than those of known c-type cytochromes from various sources. The optimum pH and temperature of the peroxidase activity were about 10.0 and 70 degrees C, respectively. The peroxidase activity is stable in the pH range of 6.0 to 10.8 (30 degrees C, 1-h treatment), and at temperatures up to 80 degrees C (pH 8.5, 20-min treatment). The heme content was determined to be 1 heme per subunit. The amino acid sequence of the cytochrome c showed high homology with those of the c-type cytochromes from Bacillus subtilis and Bacillus sp. PS3. The catalase-peroxidase showed high catalase activity and considerable peroxidase activity, the specific activities being 55,000 and 0.94 micromol/min/mg, respectively. The optimum pH and temperature of the peroxidase activity were in the range of 6.4 to 10.1 and 60 degrees C, respectively. The catalase-peroxidase showed a lower K(m) value (0.67 mM) as to H(2)O(2) than known catalase-peroxidases.     

Thermochromism of heme adducts of Glycera hemoglobin and some other monomeric heme proteins

The thermally induced difference spectra of myoglobin (Mb) and Glycera dibranchiata hemoglobin (Hbm) derivatives and of cytochrome-c were recorded between 4 degrees and 30 degrees C in the 390-750 nm range. Thermodynamic parameters were estimated and upper and lower temperature limiting spectra were deduced for the various heme protein derivatives' equilibria. The effective iron d-electron population divides the hemes broadly into two different groups of behavior type. In the first group, Hbm(III)N3, Hbm(III), Mb(III)(H2O), and Cytc(III) show equilibria between two spin states. The weakest coupling between the heme and the globin occurs among the second group, for Hbm(II)CO and Mb(II)CO, which in the higher temperature limit undergoes averaging of the carbonyl tilt, while an axially elongated geometry is probably accessed for Hbm(II)NO and Mb(II)NO. Examples of the less common situation of increased absorption intensity and/or low-spin states at higher temperature were found in both groups. In the case of the methyl thioglycolate low-spin adducts of Hbm(III), an acid/base equilibrium involving thioglycolate deprotonation occurs. Apparent enthalpy-entropy compensation is exhibited by all these heme derivatives, and it is suggested that the delta H degrees and delta S degrees values relate to the intimacy of coupling between the heme structure and the solvent-dependent microconformation of the globin.     

Solution 1H NMR investigation of the seating and rotational "hopping" of centrosymmetric etioheme-I in myoglobin: effect of globin origin and its oxidation/spin state on heme dynamics

Solution 1H NMR spectroscopy was used to investigate the heme active-site structure and dynamics of rotation about the Fe-His bond of centrosymmetric etioheme-I reconstituted into sperm whale and horse myoglobin (Mb). Comparison of the NOESY cross-peak pattern and paramagnetic relaxation properties of the cyanomet complexes confirm a heme pocket that is essentially the same as Mb with either native protoheme or etioheme-I. Dipolar contacts between etioheme and the conserved heme pocket residues establish a unique seating of etioheme that conserves the orientation of the N-Fe-N vector relative to the axial His plane, with ethyl groups occupying the vinyl positions of protoheme. Saturation transfer between methyls on adjacent pyrroles in etioheme-reconstituted horse Mb in all accessible oxidation/spin states reveals rotational hopping rates that decrease dramatically with either loss of ligands or reduction of the heme, and correlate qualitatively with expectations based on the Fe-His bond strength and the rate of heme dissociation from Mb. The rate of hopping for etioheme in metMbCN, in contrast to hemes with propionates, is the same in the sperm whale and horse proteins.     

The mechanism of reduction of cytochrome c as studied by pulse radiolysis.

1. The reaction of hydrated electrons with ferricytochrome c was studied using the pulse-radiolysis technique. 2. In 3.3 mM phosphate-buffer (pH 7.2), 100 mM methanol and at a concentration of cytochrome c of less than 20 muM the reduction kinetics of ferricytochrome c by hydrated electrons is a bimolecular process with a rate constant of 4.5-10-10 M-1-S-1 (21 degrees C). 3. At a concentration of cytochrome c of more than 20 muM the apparent order of the reaction of hydrated electrons with ferricytochrome c measured at 650 nm decreases due to the occurrence of a rate-determining first-order process with an estimated rate constant of 5-10-6s-1 (pH 7.2, 21 degrees C). 4. At high concentration of cytochrome c the reaction-time courses measured at 580 and 695 nm appear to be biphasic. A rapid initial phase (75% and 30% of total absorbance change at 580 and 695 nm, respectively), corresponding to the reduction reaction, is followed by a first-order change in absorbance with a rate constant of 1.3-10-5 S-1 (pH 7.2, 21 degrees C). 5. The results are interpreted in a scheme in which first a transient complex between cytochrome c and the hydrated electron is formed, after which the heme iron is reduced and followed by relaxation of the protein from its oxidized to its reduced conformation. 6. It is calculated that one of each three encounters of the hydrated electron and ferricytochrome c results in a reduction of the heme iron. This high reaction probability is discussed in terms of charge and solvent interactions. 7. A reduction mechanism for cytochrome c is favored in which the reduction equivalent from the hydrated electron is transmitted through a specific pathway from the surface of the molecule to the heme iron.     

Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structures of ferrous horse heart myoglobin complexed with nitric oxide and nitrosoethane

The interactions of nitric oxide (NO) and organic nitroso compounds with heme proteins are biologically important, and adduct formation between NO-containing compounds and myoglobin (Mb) have served as prototypical systems for studies of these interactions. We have prepared crystals of horse heart (hh) MbNO from nitrosylation of aqua-metMb crystals, and we have determined the crystal structure of hh MbNO at a resolution of 1.9 A. The Fe-N-O angle of 147 degrees in hh MbNO is larger than the corresponding 112 degrees angle previously determined from the crystal structure of sperm whale MbNO (Brucker et al., Proteins 1998;30:352-356) but is similar to the 150 degrees angle determined from a MS XAFS study of a frozen solution of hh MbNO (Rich et al., J Am Chem Soc 1998;120:10827-10836). The Fe-N(O) bond length of 2.0 A (this work) is longer than the 1.75 A distance determined from the XAFS study and suggests distal pocket influences on FeNO geometry. The nitrosyl N atom is located 3.0 A from the imidazole N(epsilon) atom of the distal His64 residue, suggesting electrostatic stabilization of the FeNO moiety by His64. The crystal structure of the nitrosoethane adduct of ferrous hh Mb was determined at a resolution of 1.7 A. The nitroso O atom of the EtNO ligand is located 2.7 A from the imidazole N(epsilon) atom of His64, suggesting a hydrogen bond interaction between these groups. To the best of our knowledge, the crystal structure of hh Mb(EtNO) is the first such determination of a nitrosoalkane adduct of a heme protein.     

Hemes and hemeproteins, 2: The pH-dependent equilibria of microperoxidase-8 and characterization of the coordination sphere of Fe(III)

Titration of the monomeric heme octapeptide from horse heart cytochrome c, microperoxidase-8 (MP-8) from pH 1 to pH 13 in 20% (v/v) methanol-water solutions, mu = 0.1, at 25 degrees C shows three reversible concentration-independent pKs (4.43 +/- 0.09; 8.90 +/- 0.03; 10.48 +/- 0.09) which are ascribed to successive proton loss from the conjugate acid of His (and its coordination to Fe(III)), bound H2O, and from bound His to form an imidazolate complex, respectively. The equilibrium constant for coordination of imidazole between pH 5.5 and 7.0 is independent of pH (logK = 4.45) which proves that His-18 is coordinated to Fe(III) in aqueous solution.     

Heme reduction by intramolecular electron transfer in cysteine mutant myoglobin under carbon monoxide atmosphere

Human myoglobin (Mb) possesses a unique cysteine (Cys110), whereas other mammalian Mbs do not. To investigate the effect of a cysteine residue on Mb, we introduced cysteine to various sites on the surface of sperm whale Mb (K56C, V66C, K96C, K102C, A125C, and A144C) by mutation. The cysteines were inserted near the end of alpha-helices, except for V66C, where the cysteine was introduced in the middle of an alpha-helix. Reduction of the heme was observed for each mutant metMb by incubation at 37 degrees C under carbon monoxide atmosphere, which was much faster than reduction of wild-type metMb under the same condition. Heme reduction did not occur significantly under nitrogen or oxygen atmospheres. The rate constant for heme reduction increased for higher mutant Mb concentration, whereas it did not change significantly when the CO concentration was reduced from 100% CO to 50% CO with 50% O(2). The similarity in the rate constants with different CO concentrations indicates that CO stabilizes the reduced heme by coordination to the heme iron. SDS-PAGE analysis showed that mutant Mb dimers were formed by incubation under CO atmosphere but not under air. These dimers were converted back to Mb monomers by an addition of 2-mercaptoethanol, which showed formation of a Mb dimer through a disulfide bond. The rate constant decreased in general as the heme-cysteine distance was increased, although V66C Mb exhibited a very small rate constant. Since V66 is placed in the middle of an alpha-helix, steric hindrance would occur and prevent formation of a dimer when the cysteine residues of two different V66C Mb molecules interact with each other. The rate constants also decreased for K56C and A144C Mbs presumably because of the electrostatic repulsion during dimer formation, since they are relatively charged around the inserted cysteine.