Light absorption, electron paramagnetic resonance and resonance Raman characteristics of nitridochromium(V) protoporphyrin-IX and its reconstituted hemoproteins.

A surprisingly stable complex of the photolyzed product of azidochromium(III)protoporphyrin-IX was prepared and examined by light absorption, electron paramagnetic resonance (EPR) and resonance Raman spectroscopies. The characteristic EPR spectrum for this complex was consistent with a nitridochromium(V)-porphyrin complex which was two oxidation equivalents above the resting Cr(III) complex. The Cr(V)-N stretching mode was observed at 1010 cm-1 by resonance Raman spectroscopy. A simple diatomic harmonic oscillation model gave a force constant of 6.7 mdyn/A for the Cr(V)-N bond, in the region characteristic for the metal-nitrogen triple bond. Nitridochromium(V) protoporphyrin-IX reconstituted myoglobin and cytochrome c peroxidase were prepared for the first time. The nitridochromium(V)-porphyrins in these apo-proteins were unstable in contrast with the protein-free chromium(V)porphyrin. Upon irradiation of the azide complexes of the chromium(III) protoporphyrin-IX reconstituted myoglobin and cytochrome c peroxidase with ultraviolet light aerobically at room temperature, the characteristic optical and EPR spectra for nitridochromium(V) derivatives were observed. The optical spectra of these photo-induced products were different from those of the nitridochromium(V) protoporphyrin-IX reconstituted hemoproteins. The electrochemical structures of the unusual metalloporphyrin seemed to be modulated by the heme surrounding amino acid residues.     

The reactions of myoglobin, normal adult hemoglobin, sickle cell hemoglobin and hemin with hydroxyurea

The kinetics of the reaction of hydroxyurea (HU) with myoglobin (Mb), hemin, sickle cell hemoglobin (HbS), and normal adult hemoglobin (HbA) were determined using optical absorption spectroscopy as a function of time, wavelength, and temperature. Each reaction appeared to follow pseudo-first order kinetics. Electron paramagnetic resonance spectroscopy (EPR) experiments indicated that each reaction produced an FeNO product. Reactions of hemin and the ferric forms of HbA, HbS, and myoglobin with HU also formed the NO adduct. The formation of methemoglobin and nitric oxide-hemoglobin from these reactions may provide further insight into the mechanism of how HU benefits sickle cell patients.     

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.     

New transient species of sperm whale myoglobin in photodissociation of dioxygen from oxymyoglobin

We carried out the flash photolysis of oxy complexes of sperm whale myoglobin, cobalt-substituted sperm whale myoglobin, and Aplysia myoglobin. When the optical absorption spectral changes associated with the O2 rebinding were monitored on the nanosecond to millisecond time scale, we found that the transient spectra of the O2 photoproduct of sperm whale myoglobin were significantly different from the static spectra of deoxy form. This was sharply contrasted with the observations that the spectra of the CO photoproduct of sperm whale myoglobin and of the O2 photoproducts of cobalt-substituted sperm whale myoglobin and Aplysia myoglobin are identical to the corresponding spectra of their deoxy forms. These results led us to suggest the presence of a fairly stable transient species in the O2 photodissociation from the oxy complex of sperm whale myoglobin, which has a protein structure different from the deoxy form. We denoted the O2 photo-product to be Mb*. In the time-resolved resonance Raman measurements, the nu Fe-His mode of Mb* gave the same value as that of the deoxy form, indicating that the difference in the optical absorption spectra is possibly due to the structural difference at the heme distal side rather than those of the proximal side. The structure of Mb* is discussed in relation to the dynamic motion of myoglobin in the O2 entry to or exit from the heme pocket. Comparing the structural characteristics of several myoglobins employed, we suggested that the formation of Mb* relates to the following two factors: a hydrogen bonding of O2 with the distal histidine, and the movement of iron upon the ligation of O2.     

Absorption spectra indicate conformational alteration of myoglobin adsorbed on polydimethylsiloxane.

To assess the effects of adsorption on protein structure, ultraviolet optical absorption spectra of myoglobin (Mb) bound to polydimethylsiloxane (PDMS) were measured. A flow cell, which enabled adsorption under controlled hydrodynamic conditions, was used in conjunction with a conventional spectrophotometer to obtain the spectra. Adsorption to PDMS reduced significantly the absorbance in the Soret region of the Mb spectrum, whereas the spectrum in the region near 280 nm was essentially unaffected. This result showed that disruption of the native structure of Mb occurs following interaction with PDMS. Furthermore, the change in the absorption spectrum may indicate loss of heme from the heme pocket of the adsorbed protein. Mb structure was altered from its solution configuration within fifteen min of contact with the surface. Exchange of adsorbed Mb with Mb in solution had little or no effect on the absorption spectrum of the surface-confined protein, indicating that exchange occurs only between conformationally altered species or between native species.     

Proton NMR study of the myoglobin reconstituted with meso-tetra(n-propyl)hemin

Spectrophotometric titration of meso-tetra(n-propyl)hemin with sperm-whale apomyoglobin revealed their 1:1 complex formation. The purified reconstituted metmyoglobin bound with an equal molar amount of CN- and the second CN- ligation was not evidenced, suggesting that the hemin is not loosely attached to the globin surface, but incorporated into the heme pocket. The hyperfine-shifted proton NMR spectrum of the deoxy myoglobin revealed the proximal imidazole NH resonance at 85.1 ppm to indicate the formation of the Fe-N(His-F8) bond. The eight pyrrole protons of the hemin of myoglobin in the absence of external ligand were observed as a single peak at -16 ppm. This indicates the electronic symmetry of the hemin and the low-spin configuration of the heme iron. The pyrrole-proton NMR patterns of the cyanide and deoxy myoglobins were found to be remarkably temperature-dependent, which was consistently explained in terms of the free rotation of the prosthetic group. The NMR results suggest that introduction of meso-tetra(n-propyl)hemin totally disrupts the highly stereospecific heme-globin contacts, making the prosthetic group mobile in the heme cavity.     

Dynamics of heme in hemoproteins: proton NMR study of myoglobin reconstituted with iron 3-ethyl-2-methylporphyrin

The asymmetric 3-ethyl-2-methylporphyrin iron complex was synthetized and inserted into apomyoglobin. UV-visible spectroscopic studies demonstrated the capacity of iron to coordinate different exogenous axial ligands in ferrous and ferric forms. The position of synthetic heme into the hydrophobic pocket of the reconstituted myoglobin was investigated by ((1))H NMR spectroscopy. In absence of exogenous ligand, signals of the synthetic prosthetic group were not detected, suggesting a rotational disorder of the synthetic porphyrin into the heme pocket. This direct interconversion behavior is favored since site-specific interactions between the poorly substituted heme and protein in the chiral hydrophobic cavity were weak. Complexion of cyanide to the iron allowed to quench partially the heme reorientation and two interconvertible forms, around the meso-Cα-Cγ axis, were detected in solution.     

Electrostatic interactions in wild-type and mutant recombinant human myoglobins

Residue Val68 in human myoglobin has been replaced by Asn, Asp, and Glu with site-directed mutagenesis. Purified proteins were characterized by isoelectric focusing and by absorption, CD, and NMR spectroscopy. These studies demonstrated that Mb is able to tolerate substitution of the buried hydrophobic residue Val68 by Asn, Asp, and Glu. In the metaquo derivatives of the Glu and Asp mutants, the negative charge at residue 68 is stabilized by a favorable Coulombic interaction with the heme iron. In the absence of this interaction, as in the metcyano and ferrous deoxy derivatives, the relatively nonpolar protein interior cannot stabilize an isolated buried negative charge, and the carboxylate is either protonated or stabilized via a salt bridge with the nearby distal histidine. Hence in the Asp and Glu mutant proteins, both reduction and cyanide binding are accompanied by proton uptake by the protein. The apoproteins were prepared and reconstituted with the chlorophyll derivative zinc pyrochlorophyllide a. Absorption and fluorescence spectra were quite similar for wild-type and all mutant proteins reconstituted with this derivative. These results do not support the point charge model for the red shifts observed in the spectra of chlorophylls associated with photosynthetic proteins. From the pH dependence of the absorption spectrum of zinc pyrochlorophyllide a in the Glu mutant, the apparent pKa of the buried glutamate residue was estimated to be 8.9. This increase of 4.4 pH units, over the value for Glu in aqueous solution, provides a measure of the polarity of the protein interior.     

Modification of the distal histidyl imidazole in myoglobin to N-tetrazole-substituted imidazole and its effects on the heme environmental structure and ligand binding properties.

The mechanism of N-tetrazole ring formation at the distal histidyl imidazole of sperm whale myoglobin (Mb) has been studied by nitrogen-15 (15N) NMR spectroscopy by utilizing 15N-labeled cyanogen bromide (BrCN) and azide ion (N3-). The 15N-NMR spectrum of BrC15N-modified Mb + N3- afforded two hyperfine-shifted 15N resonances, both of which are identical with the resonance positions of two of the three 15N resonances for BrCN-modified Mb + 15NN2-. This unusual spectral feature is due to the formation of the N-tetrazole ring attached to the distal histidyl imidazole and the scrambling of the labeled nitrogen originated from N3- or BrCN over the tetrazole ring upon coordination to the ferric heme iron. The ferric iron-bound N-tetrazole ring comes off upon reduction to the ferrous state, and the stable CO complex of tetrazole-modified Mb (tetrazole-Mb) is formed. Electronic absorption and 1H-NMR spectra of deoxy and carbonmonoxy forms of tetrazole-Mb are slightly altered from those of native Mb by the modification, while the most significant effect is exerted on the C-O stretching frequency of iron-bound CO. The C-O stretching band for tetrazole-MbCO is observed at 1966 cm-1 in contrast to 1945 cm-1 for native MbCO, suggesting that the geometry of iron-bound CO in tetrazole-Mb is relatively upright which is characteristic of the "open" conformer. This result corresponds to the 15-fold increase of the CO association rate constant by the N-tetrazole modification of the distal His. The oxy form of tetrazole-Mb is readily autoxidized to its ferric state, indicating that hydrogen bonding between the distal His and iron-bound oxygen is essential for stable O2 binding to the heme iron.     

Effects of formylation of vinyl side chains of heme on optical and ligand binding properties of horse heart ferric myoglobin.

Effects of substitution of vinyl groups of hemin with formyl groups on the optical and ligand binding properties of horse heart ferric myoglobin were investigated. The peak positions as well as the line shapes of the absorption spectra of the ferric derivatives of three kinds of formylmyoglobin, 2-vinyl-4-formyl-, 2-formyl-4-vinyl-, and 2,4-diformylmyoglobins depend on the number and the position of the formyl groups. Absorption maxima in the Soret region of the acid forms of these ferric formylmyoglobins in 0.1 M potassium phosphate buffer, pH 6.0, at 20 degrees were 415.2, 422, and 429 nm, respectively. The acid forms of these formylmyoglobins exhibit absorption spectra of the mixture of high- and low spin states at ambient temperature. Since proto-, deutero- and mesomyoglobins have a high spin state under the same condition, the increase of the low spin iron in these formylmyoglobins may be due to the strong electron withdrawal by the formyl groups toward the periphery of the porphyrin ring. The affinities of these ferric formylmyoglobins and protomyoglobin for N3-, F-, OCN-, and SCN- increased in the order of proto-, monoformyl-monovinyl-, 2,4-diformyl-myoglobin, which corresponds to the increasing order of electron-withdrawing power of the porphyrin side chains. The pKa values of the acid-alkaline transition decreased in the same order. Although the ferric forms of the two isomeric monoformyl-monovinylmyoglobins exhibited different optical spectra, the dissociation constants of the complexes of these isomers for various ligands were similar to each other. The pKa values of the acid-alkaline transition were also similar. These results indicate that affinities of ferric myoglobin for ligands, in contrast to those of the ferrous form for oxygen and carbon monoxide (Sono, M., and Asakura, T. (1975) J. Biol. Chem. 250, 5527-5232 and Sono, M., Smith, P.D., McCray, J.A., and Asakura, T. (1976) J. Biol. Chem 251, 1418-1426), are not affected by the position of modifications at the two vinyl groups, but are determinedby the number of the formyl groups and that two vinyl groups at position 2 and 4 are equivalent in the binding of various ligands by ferric myoglobin. The electron density of the ferric iron appears to be similar for the two isomeric monoformyl-monovinylmyoglobins.     

Functional effects of heme orientational disorder in sperm whale myoglobin

The optical absorption and ligand binding properties of newly reconstituted sperm whale myoglobin were examined systematically at pH 8, 20 degrees C. The conventional absorbance and magnetic circular dichroism spectra of freshly reconstituted samples were identical to those of the native protein. In contrast, reconstituted azide or CO myoglobin initially exhibited less circular dichroism in the Soret wavelength region than native myoglobin. These data support the theory proposed by La Mar and co-workers (La Mar, G. N., Davis, N. L., Parish, D. W., and Smith, R. M. (1983) J. Mol. Biol. 168, 887-896) that protoheme inserts into apomyoglobin in two distinct orientations. The equilibrium and kinetic parameters for O2 and CO binding to newly reconstituted myoglobin were observed to be identical to those of the native protein. Thus, the orientation of the heme group has no effect on the physiological properties of myoglobin. This result is in disagreement with the preliminary report of Livingston et al. (Livingston, D. J., Davis, N. L., La Mar, G. N., and Brown, W. D. (1984) J. Am. Chem. Soc. 106, 3025-3026) which suggested that the abnormal heme conformation exhibited a 10-fold greater affinity and association rate constant for O2 binding. Significant kinetic heterogeneity was observed only for long-chain isonitrile binding to newly reconstituted myoglobin, and even in these cases, the rate constants for the abnormal and normal heme conformations differed by less than a factor of 4.     

Spectroscopic and interfacial properties of myoglobin/surfactant complexes

The complexes of horse myoglobin (Mb) with the anionic surfactant sodium dodecyl sulfate (SDS), and with the cationic surfactants cetyltrimethylammonium chloride (CTAC) and decyltrimethylammonium bromide (DeTAB), have been studied by a combination of surface tension measurements and optical spectroscopy, including heme absorption and aromatic amino acid fluorescence. SDS interacts in a monomeric form with Mb, which suggests the existence of a specific binding site for SDS, and induces the formation of a hexacoordinated Mb heme, possibly involving the distal histidine. Fluorescence spectra display an increase of tryptophan emission. Both effects point to an increased protein flexibility. SDS micelles induce both the appearance of two more heme species, one of which has the features of free heme, and protein unfolding. Mb/CTAC complexes display a very different behavior. CTAC monomers have no effect on the absorption spectra, and only a slight effect on the fluorescence spectra, whereas the formation of CTAC aggregates on the protein strongly affects both absorption and fluorescence. Mb/DeTAB complexes behave in a very similar way as Mb/CTAC complexes. The surface activity of the different Mb/surfactant complexes, as well as the interactions between the surfactants and Mb, are discussed on the basis of their structural properties.     

Acid and alkaline forms of the higher oxidation state of kangaroo, horse, and sperm whale myoglobin.

Myoglobin(IV), the derivative of myoglobin at the formal oxidation state IV, prepared from kangaroo (Megaleia rufa), horse, or sperm whale myoglobin, when cooled to liquid nitrogen temperature, assumes acid and alkaline forms with different optical spectra. The essential features of the optical spectra of the acid forms are the same as those of leghemoglobin(IV) and are very similar to those of optical spectra of the red higher oxidation states of catalases and peroxidases. This shows that the configuration of the heme iron is the same throughout these compounds. That configuration is believed to be Fe(IV) in a porphyrin environment. The optical spectra of alkaline mammalian myoglobin(IV), like that of alkaline leghemoglobin(IV), resemble those of the alkaline low spin ferric proteins. Kangaroo myoglobin(IV) may be prepared by reaction of ferrous myoglobin with hydrogen peroxide. The acid forms of myoglobin(IV) are conveniently prepared by cooling solutions in borate buffers, initially pH 8.3, to liquid nitrogen temperature. At this temperature borate buffers become acidic.     

19F NMR study of protein-induced rhombic perturbations on the electronic structure of the active site of myoglobin

 A novel C ₂-symmetric ring-fluorinated hemin, 13,17-bis(2-carboxyethyl)-2,8,12,18-tetramethyl-3,7-difluoroporphyrinatoiron(III), has been synthesized and was incorporated into sperm whale apomyoglobin to investigate protein-induced rhombic perturbations on the electronic structure of the active site of myoglobin (Mb) using ¹⁹F NMR spectroscopy. NMR signals for ¹⁹F atoms introduced as substituents on the present heme in ferrous low-spin and high-spin and ferric low-spin complexes have been observed and their shifts sharply reflect not only the electronic nature of the heme iron, but also in-plane asymmetry of the heme electronic structure. The two-fold symmetric electronic structure of the ring-fluorinated hemin is clearly manifested in the ¹⁹F and ¹H NMR spectra of its dicyano complex. The chemical equivalence of the two fluorine atoms of the heme is removed in the active site of myoglobin and the splitting of the two ¹⁹F NMR signals provides a quantitative probe for characterizing the rhombic perturbation of the heme electronic structure induced by the heme-protein interaction. The in-plane asymmetry of heme electronic structures in carbonmonoxy and deoxy Mbs have been analyzed for the first time on the basis of the shift difference between the two ¹⁹F NMR signals of the heme and is interpreted in terms of iron-ligand binding and/or the orbital ground state of the heme. A potential utility of ¹⁹F NMR, combined with the use of a symmetric fluorinated hemin, in characterizing the heme electronic structure of myoglobin in a variety of iron oxidation, spin, and ligation states, is presented. Received: 23 December 1999 / Accepted: 3 April 2000     

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)     

Haem-binding-site heterogeneity and haem Cotton effects of Glycera dibranchiata monomeric haemoglobins.

The five major components of the monomeric haemoglobin from Glycera dibranchiata were separated and characterized by absorption spectroscopy, isoelectric focusing, azide-binding affinities and nitrosyl autoreduction kinetics. The differences found among the components are discussed in terms of haem-pocket variations. In addition, the Fourier-transform i.r. spectra of pooled monomeric haemoglobin carbonyl (HbmCO) and the major component carbonyl are reported. The c.d. spectra of the carbonyl and azide derivatives of the five components are compared and found to be similar. The c.d. spectra of myoglobin(II) carbonyl [Mb(II)CO] and of apomyoglobin (apoMb) reconstituted with a symmetric synthetic iron porphyrin carbonyl, meso-tetra-(p-carboxyphenyl)porphinatoiron(II) carbonyl [TCPPFe(II)CO], are compared with the c.d. spectra of pooled HbmCO and its TCPPFe(II)CO analogue. HbmTCPPFe(II)CO shows a negative Soret c.d. band whereas MbTCPPFe(II)CO produces both a negative and a positive Soret c.d. band. Displacement of the symmetric porphyrin by 8-anilinonaphthalene-1-sulphonate and the resulting fluorescence emission are reported.     

Time-resolved circular dichroism and absorption studies of the photolysis reaction of (carbonmonoxy)myoglobin.

Time-resolved circular dichroism (TRCD) and absorption spectroscopy are used to follow the photolysis reaction of (carbonmonoxy)myoglobin (MbCO). Following the spectral changes associated with the initial loss of CO, a subtle change is observed in the visible absorption spectrum of the Mb product on a time scale of a few hundred nanoseconds. No changes are seen in the CD spectrum of Mb in the visible and near-UV regions subsequent to the loss of CO. The data suggest the existence of an intermediate found after ligand loss from MbCO that is similar in structure to the final Mb product.     

Proton NMR study of myoglobin reconstituted with 3, 7-diethyl-2, 8-dimethyl iron porphyrin: remarkable influence of peripheral substitution on heme rotation

The iron complex of 3,7-diethyl-2,8-dimethylporphyrin was incorporated into horse heart apomyoglobin to investigate the influence of peripheral substitution on artificial heme rotation. The hyperfine-shifted 1H NMR spectrum of the reconstituted deoxymyoglobin (rMb) revealed the proximal imidazole N-H resonance at 82.5 ppm to indicate the formation of the Fe--N (His93) bond. The pyrrole-protons of the hemin of myoglobin in the absence of external ligand appeared as four resonances between -10 and -18 ppm, indicating a mainly low-spin ferric hemin, with a ligated distal histidine (His64). This also indicates the lost of the symmetry of the hemin, according to an absence of free rotation of the prosthetic group. The 1H NMR spectrum of reconstituted rMbCO revealed a set of four pyrrole-protons and a set of four meso-protons. Accordingly, the prosthetic group without acid side chains interacts specifically with the surrounding globin showing a unique heme orientation in the 1H NMR time-scale, despite the presence of only four alkyl substituents on the porphine ring. This also suggests that two ethyl groups are large enough to avoid the free rotation movement of the heme.     

An ESR study of nitrosyl-Aplysia brasiliana myoglobin and nitrosyl annelidae Glossoscolex paulistus erythrocruorin

The nitrosyl derivatives of Annelidae Glossoscolex paulistus hemoglobin (an earth worm erythrocruorin (Ec AGp)) and Aplysia brasiliana myoglobin (Mb Apb) are studied using ESR spectroscopy. These two proteins have a quite similar ESR spectra at 100 K, but a different temperature behaviour. The temperature dependence of the nitrosyl Mb Apb spectrum is in good agreement with the Boltzmann distribution. In the case of nitrosyl-Ec AGp, the results are explained by the existence of two types of spectrum in thermodynamic equilibrium, with delta H = 9.08 kJ/mol, delta S = 47.15 J/mol and T1/2 = 193 K. There is a great similarity of the nitrosyl-Ec AGp spectra with those reported for elephant myoglobin, suggesting the presence of the same heme environment with a glutamine residue in the distal site. The pH dependence of the spectrum of nitrosyl-Mb Apb shows that the affinity of nitrosyl binding is higher at high pH (7.3) than at low pH (4.6). The ESR parameters are the same for these two pH values.     

Site-specific covalent attachment of heme proteins on self-assembled monolayers

Naturally occurring hemin cofactor has been functionalized to introduce two terminal alkyne groups. This modified hemin has been successfully covalently attached to mixed self-assembled monolayers of alkanethiols and azide-terminated alkanethiols on gold electrodes using a Cu(I)-catalyzed 1,3-cycloaddition reaction. However these hemin-modified electrodes could not be used to reconstitute apomyoglobin on gold electrodes owing to the hydrophobicity of the alkane thiol self-assembled monolayer. Modification of existing techniques allowed covalent attachment of alkyne-terminated electroactive species onto mixed monolayers of azidothiols and carboxylatoalkanethiols on electrodes using the same Cu(I)-catalyzed 1,3-cycloaddition reaction. Apomyoglobin could be reconstituted using the hemin covalently attached to these hydrophilic electrodes. The electrochemical data, UV-vis absorption data, surface-enhanced resonance Raman spectroscopy data, and atomic force microscopy data indicate the presence of these modified myoglobin proteins on these electrodes. The direct attachment of the heme cofactor of these modified myoglobin proteins to the electrode allows fast electron transfer to the heme center from the electrode and affords efficient O(2)-reducing bioelectrodes under physiological conditions.