Short communication

Analysis of a thermal equilibrium phenomenon between high-spin and low-spin states of ferrimyoglobin azide

The physicochemical properties of a series of the six-coordinated iron(III) diazaporphyrinate complexes, [Fe(DAzP)L₂]^± where the axial ligands (L) are DMAP, CN⁻, HIm, Py, 3-ClPy, 4-CNPy, 3-CNPy, 3,5-Cl₂Py, THF, have been examined by combined analyses of NMR, EPR, Mössbauer spectrometry, and SQUID magnetometry. The solution and solid-state studies clarified that the complexes with 3-ClPy, 4-CNPy, 3-CNPy, 3,5-Cl₂Py are in the less common spin-crossover process between S = 3/2 and S = 1/2 during the temperature change. The results clearly indicate that the complexes showed the switching behaviors of the electronic structure or the magnetic properties responsive to the external stimuli.

The acetylation of the hemeundecapeptide prepared by proteolysis of cytochrome c yields a species di (N-acetyl)-microperoxidase-ll, NAcMP11, that is monomeric in aqueous solution at least for concentrations below 20 μM, in contrast to MP11 itself, which aggregates because of intermolecular coordination of Fe (III) by the N-terminal amino group or the amino group of the side chain of Lys-13. The present report complements a report by Peterson and co-workers on the preparation and properties of NAcMPl 1 (Inorg. Chem. 35 (1996) 6885). We show that NAcMP11 has six spectroscopically observable pH-dependent transitions at 1.90±0.03, 3.37±0.01, 4.6±0.1, 5.4±0.03, 9.56±0.01 and 12.4±0.03. The first is probably due to displacement of one of two H₂O molecules from the coordination sphere of Fe(III) by the C-terminal Glu-21 carboxylate (giving the axial ligand combination RCOO⁻/H₂O); as the pH is raised, His-18 is deprotonated and coordinates the metal (His/H₂O). The next two transitions are due to ionization of heme propionic acid groups; the penultimate is caused by the ionization of Fe(III)-bound H₂O (His/OH⁻); and the final transition is from ionization of His-18 to form a histidinate (His⁻/OH⁻). The EPR spectrum of NAcMP11 at pH 0.7 is consistent with a mixture of a di(aqua) and a mono(aqua) species. Both the aqua complex of NAcMPll (at pH 7.6) and the hydroxo complex (at pH 11.0) are in equilibrium between a quantum-mechanically admixed spin state (S = 3/2, 5/2) and a low-spin state (S= 1/2). The crystal field parameters of the two complexes (which are similar) as derived from the EPR spectrum are reported. The EPR spectrum at pH 13.8 shows that the hydroxo-histidinate complex of NAcMPl 1 undergoes a slow reaction, possibly to form a di(hydroxo) complex with displacement of the histidinate ligand, or a dimerization with the histidinate acting as bridging ligand. The coordinated H₂O molecule in NAcMP11 is readily replaced by an exogenous ligand, and binding constants for coordination of cyanide, imidazole, azide and chloride are reported. NAcMP-11 is shown to display similar physical and chemical properties to the analogous octapeptide, NAcMP-8, but is easier to prepare; this makes NAcMP-11 a useful alternative model for the hemoproteins.

The temperature dependence of the dissociation of cyanide (CN⁻) from cyanomyoglobin (Mb(II)CN), the cyanide complex of myoglobin, has been studied by cyclic voltammetry. Direct electrochemical reduction of cyanometmyoglobin initiates the dissociation of the cyanide ligand. The temperature dependence of the heterogeneous electron transfer kinetics, the kinetics of cyanide dissociation, and the formal potential for Mb(III)CN have been determined over the temperature range of 10–50°C. The latter information has been used to determine the reaction center entropy change (ΔS_rc) of this reaction to be −90±5 J mol⁻¹ K⁻¹. The water complex of myoglobin, aquometmyoglobin (Mb(III)H₂O), was also studied and the reaction center entropy for this system was determined to be −40±4 J mol⁻¹ K⁻¹. Differential scanning calorimetry was also used to determine the transition temperature and enthalpy changes for each component of these reactions. The results given here provide an indirect model for the in vivo reaction of the oxymyoglobin complex (Mb(II)O₂), that is, the dissociation of dioxygen from the reduced form of myoglobin.

NMR studies of paramagnetic haemoproteins are reviewed with special emphasis on characterization of both structural and dynamic properties of haem active site. In the past decade, the development of NMR methodologies for detecting the connections between hyperfine shifted signals has contributed greatly to establishing systematic and reliable strategies for the signal assignments of paramagnetic haemoproteins. The use of reconstituted haemoprotein is a characteristic of the study of b-type haemoproteins and NMR studies on reconstituted myoglobins and haemoglobins are described in some detail. Additionally, nonequivalence in haem electronic structure between the two different subunits in human adult haemoglobin enables one to characterize the individual subunits in intact tetramers using NMR, and the advantages in studying tetrameric haemoglobin by N M R are also described.

This chapter discusses the recent advances in resonance Raman spectroscopy of ligand binding (exogenous and endogenous) to hemoproteins, and heme model complexes. Some aspects of Raman spectroscopic techniques essential for ligand binding studies are presented first. Important bases for vibrational data analysis and interpretation are also described. The exogenous ligands discussed here include carbon monoxide, cyanide, nitric oxide, dioxygen fluoride, and azide; endogenous ligands include histidine, tryosine, and cysteine. The power of resonance Raman spectroscopy for ligand binding studies lies in its ability to detect the metal-ligand vibrations in both model complexes and hemoproteins. The structural data from model complexes as determined by X-ray diffraction can then be employed to deduce the ligand structure in hemoproteins, where the X-ray crystallographic analysis is less accurate. Infrared spectroscopy is capable of detecting the metal-ligand vibrations in model compounds in crystals or in nonaqueous solution; yet it is difficult, if not impossible, to obtain similar information in hemoproteins because of strong water absorption and the interference due to other vibrational modes from the protein matrix. The sensitivity and selectivity of resonance Raman techniques permit the detection of metal-ligand vibrations in aqueous solution as low as ∼10^-5 M, compared to the ∼10^-3 M concentration used in infrared studies of internal ligand vibrations such as v (C—O), v (O—O), v (N—O), v (N=N=N) and v (C≡N).