A magnetic study of acidic ferric hemoglobin

In this article, we have extensively studied and discussed the magnetic properties of acidic ferric hemoglobin and its isolated chains. The magnetic susceptibility, EPR and optical spectra of those samples were measured in the temperature region below 77 degrees K. By the magnetic susceptibility measurements, it could be made clear that at an acidic pH value, both ferric hemoglobin and its isolated chains were constituted of a mixture of two spin states (high-spin state S = 5/2 and low-spin state S = 1/2) and the ratio of this mixture varied in each protein sample, but was independent of the temperature change below 77 degrees K. The co-existence of these two components could be ascertained by the observation of EPR spectra at liquid hydrogen temperature. Acidic ferric hemoglobin and its isolated chains exhibited the two components of EPR spectra which corresponded to their magnetic susceptibility, and it was found that the relaxation time of the low-spin state was longer than that of the high-spin state. The low-spin component of EPR spectra was almost undetectable at liquid nitrogen temperature. The three principal g values of this low-spin were gz = 2.80, gy = 2.20, and gx = 1.70. At alkaline pH values these low-spin components and the high-spin component of EPR spectra were displaced by the different low-spin spectra which corresponded to the ferric hemoglobin-hydroxide complex. It seems that the magnetic properties of the high-spin component are the same as the acidic ferric myoglobin, and the fine structure of the iron ion also seems to be same. Optical spectroscopy also gave similar magnetic properties which corresponded to the magnetic measurements.     

Angular dependence of dipole-dipole-Curie-spin cross-correlation effects in high-spin and low-spin paramagnetic myoglobin

The ¹⁵N-HSQC spectra of low-spin cyano-met-myoglobin and high-spin fluoro-met-myoglobin were assigned and dipole-dipole-Curie-spin cross-correlated relaxation rates measured. These cross-correlation rates originating from the dipolar ¹H-¹⁵N interaction and the dipolar interaction between the ¹H and the Curie spin of the paramagnetic center contain long-range angular information about the orientation of the ¹H-¹⁵N bond with respect to the iron-¹H vector, with information measurable up to 11 Å from the metal for the low-spin complex, and between 10 to 25 Å for the high-spin complex. Comparison of the experimental data with predictions from crystal structure data showed that the anisotropy of the magnetic susceptibility tensor in low spin cyano-met-myoglobin significantly influences the cross-correlated dipole-dipole-Curie-spin relaxation rates.     

Optical investigation of spin-crossover in cobalt(II) bis-terpy complexes

The spin transition of the [Co(terpy)₂]²⁺ complex (terpy = 2,2′:6′,2″-terpyridine) is analysed based on experimental data from optical spectroscopy and magnetic susceptibility measurements. The single crystal absorption spectrum of [Co(terpy)₂](ClO₄)₂ shows an asymmetric absorption band at 14 400 cm⁻¹ with an intensity typical for a spin-allowed d-d transition and a temperature behaviour typical for a thermal spin transition. The single crystal absorption spectra of suggest that in this compound, the complex is essentially in the high-spin state at all temperatures. However, the increase in intensity observed in the region of the low-spin MLCT transition with increasing temperature implies an unusual partial thermal population of the low-spin state of up to about 10% at room temperature. Finally, high-spin → low-spin relaxation curves following pulsed laser excitation for [Co(terpy)₂](ClO₄)₂ dispersed in KBr discs, and as a comparison for the closely related [Co(4-terpyridone)₂](ClO₄)₂ spin-crossover compound are given.     

Surface enhanced resonance Raman scattering as a probe of the spin state of structurally related cytochromes P-450 from rat liver

Surface enhanced resonance Raman scattering (SERRS) was observed from structurally related drug-induced rat liver cytochromes P-450 adsorbed on a silver colloid. Careful control of pH and the sequence of addition of components to the so1 is required to prevent protein denaturation at the surface due to conversion to P-450's biologically inactive form P-420 or haem loss. A low-spin P-450 (PB3a), a mixed low- and high-spin P-450 (PB3b) and a predominantly high-spin P-450 (MC1a) were investigated. Spectra recorded in the 1300-1700 cm-1 frequency region, containing the oxidation state marker v4 at 1375 cm-1 (Fe3+) and spin state markers v10 (1625 cm-1, high-spin; 1633 cm-1, low-spin) and v19 (1575 cm-1, high-spin; 1585 cm-1, low-spin) were used to differentiate between the spin states of the various forms of cytochrome P-450. As well as the established spin state marker bands, the intensity of a band at 1400 cm-1 appeared to depend on the high-spin content. Thus, with this method SERRS from silver colloids can be used to determine spin states of related cytochromes P-450 in dilute solution (10(-8)M) and may be of value in studies of protein-substrate interactions.     

Spin States of Divalent Iron in Anhydrohemoglobin

Mössbauer spectra of anhydrohemoglobin establish the existence of two ferrous iron spin states in anhydrohemoglobin. Magnetic susceptibility measurements show that anhydrohemoglobin is paramagnetic and has an effective magnetic moment per iron atom of 3.5 ± 0.1 μ_B at low temperatures. In conjunction with the susceptibility results, the Mössbauer spectra indicate that the iron in anhydrohemoglobin is distributed between high and low spin states in roughly equal amounts.     

NMR and electron-paramagnetic-resonance studies of a dihaem cytochrome from Pseudomonas stutzeri (ATCC 11607) (cytochrome c peroxidase)

A dihaem cytochrome (Mr 37 400) with cytochrome c peroxidase activity was purified from Pseudomonas stutzeri (ATCC 11 607). The haem redox potentials are far apart: one of the haems is completely ascorbate-reducible and the other is only reduced by dithionite. The coordination, spin states and redox properties of the covalently bound haems were probed by visible, NMR and electron paramagnetic resonance (EPR) spectroscopies in three oxidation states. In the oxidized state, the low-temperature EPR spectrum of the native enzyme is a complex superimposition of three components: (I) a low-spin haem indicating a histidinyl-methionyl coordination; (II) a low-spin haem indicating a histidinyl-histidinyl coordination; and (III) a minor high-spin haem component. At room temperature, NMR and optical studies indicate the presence of high-spin and low-spin haems, suggesting that for one of the haems a high-spin to low-spin transition is observed when temperature is decreased. In the half-reduced state, the component I (high redox potential) of the EPR spectrum disappears and induces a change in the g-values and linewidth of component II; the high-spin component II is no longer detected at low temperature. Visible and NMR studies reveal the presence of a high-spin ferric and a low-spin (methionyl-coordinated) ferrous state. The NMR data fully support the haem-haem interaction probed by EPR. In the reduced state, the NMR spectrum indicates that the low-potential haem is high-spin ferrous.     

Spectroscopic studies of protein-heme interactions accompanying the allosteric transition in methemoglobins

Resonance Raman, optical absorption, and circular dichroism spectroscopic techniques have been used to examine the effect of the addition of inositol hexaphosphate (IHP) to a series of carp and human methemoglobin derivatives. Markers of spin equilibrium in the high-frequency region (1450-1650 cm-1) of the resonance Raman spectrum yield high/low-spin ratios consistent with direct magnetic susceptibility measurements. Changes in the low-frequency region (100-600 cm-1) of the resonance Raman spectrum appear to correlate with the quaternary structure transition. Changes in the ultraviolet absorption spectra and the circular dichroism spectra also appear to be related to the quaternary structure change. By using the resonance Raman spin markers, we find that those derivatives of carp methemoglobin which are in spin equilibrium have a larger ratio of high-spin to low-spin populations than the corresponding derivatives of human methemoglobin. Upon the addition of IHP to the methemoglobins the spin equilibrium is shifted toward a larger high-spin population. This change in equilibrium is larger for the carp protein than for the human protein. We obtain an IHP-induced change in the free energy difference between the high-spin and low-spin states of 300 cal/mol for those human methemoglobins in which a quaternary structure change occurs and 600 cal/mol for carp methemoglobins. Our data are consistent with a quaternary structure change induced by IHP in all the carp methemoglobins studied (F-, H2O, SCN-, NO2-, N3-, and CN-) and in the F-, H2O, and SCN- derivatives of the human protein but not in the NO2-, N3-, and CN- derivatives. The Fe-CN stretching mode has been identified by isotopic substitution and found to be unchanged in frequency in carp CN- metHb when the quaternary structure is changed. On the basis of our results we conclude that the protein forces at the heme due to the addition of IHP do not significantly affect the position of the iron atom with respect to the heme plane. Rather, the changes in spin equilibrium may be caused by protein-induced changes in the orientation of the proximal histidine or tertiary structure changes in the heme pocket which affect the porphyrin macrocycle. Either of these changes, or a combination thereof, leads to changes in the iron d orbital energies and concomitant changes in the spin equilibrium.     

Interaction of human adult methemoglobin in low-spin state with inositol hexaphosphate. A proton magnetic resonance study

The hyperfine-shifted proton nuclear magnetic resonance (NMR) spectra of the low-spin complexes of human adult methemoglobin were found to be much altered by the addition of inositol hexaphosphate (IHP). The stoichiometry and pH-dependence of IHP binding, and the spin equilibrium of azide methemoglobin are parallel to those of high-spin human methemoglobin and of carp methemoglobin, both of which are proposed to be switched from the R to T states with IHP. The present NMR results show that IHP affects the structure of human methemoglobin regardless of the spin state of the heme iron, suggesting that there is no correspondence between quaternary structure and the spin state of ferric heme iron.     

Paramagnetic properties of the low- and high-spin states of yeast cytochrome c peroxidase

Here we describe paramagnetic NMR analysis of the low- and high-spin forms of yeast cytochrome c peroxidase (CcP), a 34 kDa heme enzyme involved in hydroperoxide reduction in mitochondria. Starting from the assigned NMR spectra of a low-spin CN-bound CcP and using a strategy based on paramagnetic pseudocontact shifts, we have obtained backbone resonance assignments for the diamagnetic, iron-free protein and the high-spin, resting-state enzyme. The derived chemical shifts were further used to determine low- and high-spin magnetic susceptibility tensors and the zero-field splitting constant (D) for the high-spin CcP. The D value indicates that the latter contains a hexacoordinate heme species with a weak field ligand, such as water, in the axial position. Being one of the very few high-spin heme proteins analyzed in this fashion, the resting state CcP expands our knowledge of the heme coordination chemistry in biological systems.     

Studies on the bacterial hemoglobin fromVitreoscilla

Summary Vitreoscilla contained a homodimeric bacterial hemoglobin (VtHb). The purification of this protein yielded VtmetHb which exhibited electronic and electron paramagnetic resonance (EPR) spectra, showing that it existed predominantly in a high-spin ferric form, both axial and rhombic components being present. The preparations also contained variable amounts of low-spin components. There was no evidence that these high-spin and low-spin forms were in equilibrium. The former were reducible by NADH catalyzed by the NADH-metVtHb reductase, and the latter were not. High ionic strength and high pH led to the formation of low-spin metVtHb; both treatments were reversible. Cyanide and imidazole liganded to VtHb resulted in the conversion of high-spin to low-spin ferric heme centers, each with characteristic electronic and EPR spectra. Some preparations of VtHb exhibited EPR signals consistent with a sulfur ligand bound to the ferric site. When VtHb was treated with NADH plus the reductase in the presence of oxygen, the intensity of the high-spin EPR signals decreased significantly. No reduction occurred in the absence of oxygen, suggesting a possible role for the superoxide anion. Dithionite treatment of VtHb resulted in a slow reduction, but the main product of the reaction of dithionite-reduced VtHb with oxygen was VtmetHb, not VtHbO₂. EPR spectra of whole cells ofVitreoscilla exhibited a variety of intense signals at low and high magnetic field, theg-values being consistent with the presence of high-spin ferric heme proteins, in addition to an iron-containing superoxide dismutase (FeSOD) and iron-sulfur proteins. EPR spectra of the cytosol fraction ofVitreoscilla showed the expected resonances for VtmetHb and FeSOD.Abbreviations A absorbance - DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetate - EPR electron paramagnetic resonance - HiPIP high-potential iron protein - SDS sodium dodecyl sulfate - SOD superoxide dismutase - VtHb Vitreoscilla hemoglobin - VtmetHb oxidizedVitreoscilla hemoglobin - VtHbO₂ oxygenatedVitreoscilla hemoglobin     

Redox chemistry of low-pH forms of tetrahemic cytochrome c3

Desulfovibrio vulgaris Hildenborough cytochrome c₃ contains four hemes in a low-spin state with bis-histidinyl coordination. High-spin forms of cytochrome c₃ can be generated by protonation of the axial ligands in order to probe spin equilibrium (low-spin/high-spin). The spin alterations occurring at acid pH, the associated changes in redox potentials, as well as the reactivity towards external ligands were followed by the conjunction of square wave voltammetry and UV–visible, CD, NMR and EPR spectroscopies. These processes may be used for modelling the action of enzymes that use spin equilibrium to promote enzyme activity and reactivity towards small molecules.     

Heme complexes of rabbit hemopexin,human hemopexin and human serum albumin: Electron spin resonance and Mőssbauer spectroscopic studies

The electron spin resonance (ESR) spectra of human and rabbit ferriheme-hemopexin complexes at 30^oK show an ESR absorption characterized by g_x = 1.60, g_y = 2.25 and g_z = 2.86, characteristic of low-spin ferriheme-proteins. The low-spin nature of the heme-iron in heme-hemopexin is corroborated by the observation of nuclear hyperfine splitting in M?ssbauer spectra at 4.2^oK. The similarity of the ESR spectra of ferriheme-hemopexin with those of low-spin cytochromes which coordinate heme via two histidine residues points to a similar coordination mechanism in hemopexin. In contrast, the ESR spectra of the 1:1 and 2:1 complexes of heme with human serum albumin display signals at g = 6.0 and g = 2.0, characteristic of high-spin ferrihemeproteins.     

Resonance Raman spectroscopy shows different temperature-dependent coordination equilibria for native horseradish and cytochrome c peroxidase

Resonance Raman spectra are reported for native horseradish peroxidase (HRP) and cytochrome c peroxidase (CCP) at 290, 77 and 9 K, using 406.7 nm excitation, in resonance with the Soret electronic transition. The spectra reveal temperature-dependent equilibria involving changes in coordination or spin state. At 290 K and pH 6.5, CCP contains a mixture of 5- and 6-coordinate high-spin Fe^III heme while at 9 K the equilibrium is shifted entirely to the 6-coordinate species. The spectra indicate weak binding of H₂O to the heme Pe, consistent with the long distance, 2.4 Å, seen in the crystal structure. At 290 K HRP also contains a mixture of high-spin Fe^III hemes with the 5-coordinate form predominant. At low temperature, a small 6-coordinate high-spin component remains but the 5-coordinate high-spin spectrum is replaced by another which is characteristic either of 6-coordinate low-spin or 5-coordinate intermediate spin heme. The latter species is definitely indicated by previous EPR studies at low temperature. This behavior implies that, in contrast to CCP, the distal coordination site of HRP is only partially occupied by H₂O at any temperature and that lowering the temperature significantly weakens the Fe-proximal imidazole bond. Consistent with this inference, the 77 K spectrum of reduced HRP shows an appreciable fraction of molecules having an Fe-imidazole stretching frequency of 222 cm⁻¹, a value indicating weakened H-bonding of the proximal imidazole.Resonance Roman spectroscopyHorseradish peroxidaseCytochrome c peroxidaseCoordination equilibrium     

Low-lying electronic states of the ferrous high-spin (S=2) heme in deoxy-Mb and deoxy-Hb studied by highly-sensitive multi-frequency EPR

The low-lying electronic states of the ferrous high-spin heme in deoxy-myoglobin (deoxy-Mb) and deoxy-hemoglobin (deoxy-Hb) were probed by multi-frequency electron paramagnetic resonance (MFEPR) spectroscopy. An unexpected broad EPR signal was measured at the zero magnetic field using cavity resonators at 34-122 GHz that could not be simulated using any parameter sets for the S = 2 spin Hamiltonian assuming spin quintet states in the ⁵B₂ ground state. Furthermore, we have observed novel, broad EPR signals measured at 70-220 GHz and 1.5 K using a single pass transmission probe. These signals are attributed to the ferrous high-spin heme in deoxy-Mb and deoxy-Hb. The resonant peaks shifted to a higher magnetic field with increasing frequency. The energy level separation between the ground singlet and the first excited state at the zero magnetic field was directly estimated to be 3.5 cm^− 1 for deoxy-Hb. For deoxy-Mb, the first two excited singlet states are separated by 3.3 cm^− 1 and 6.5 cm^− 1, respectively, from the ground state. The energy gap at the zero magnetic field is directly derived from our MFEPR for deoxy-Mb and deoxy-Hb and strongly supports the theoretical analyses based on the Mössbauer and magnetic circular dichroism experiments.     

Spectral properties of myeloperoxidase and its ligand complexes

The effects of ligands with various field strengths on the optical absorption spectrum of myeloperoxidase have been investigated. As is the case with other hemoproteins, the Soret peak in the optical absorption spectra at 77 K moves to longer wavelengths when strong-field ligands are present, whereas binding of such ligands as chloride and fluoride, which stabilize the high-spin state, shows the opposite effect. With a ligand of intermediate field strength, such as azide, the optical spectrum is not affected at room temperature, but lowering of the temperature results in the formation of the low-spin form of the enzyme. Similarly, in native myeloperoxidase a spin state equilibrium is found in which the low-spin state is favoured at high ionic strength and displays corresponding changes in the optical spectra. From the ligand- and the temperature-induced changes in the optical spectra of the ferric enzyme it is concluded that the band at 620-630 nm is an alpha band of the low-spin heme iron species, whereas the bands at 500 and 690 nm are probably 'charge-transfer' bands of the heme with the iron in the high-spin state.     

Synthesis, crystal structures and properties of iron(III) complexes exhibited both high-spin and low-spin states within the crystal lattice

The reaction of FeCl₃ · 6H₂O, potassium hydrotris(pyrazolyl)borate (KTp) and KSCN gives [FeTp₂][TpFe(NCS)₃] (1) and [FeTp₂]₃[Fe(NCS)₆] (2), respectively. The bond lengths and angles indicate that both complexes have [FeTp₂]⁺ cations where Fe(III) ions are in typical low-spin state, and in counter ions, [TpFe(NCS)₃]⁻ for 1 and [Fe(NCS)₆]³⁻ for 2 are both in high-spin state. Variable temperature magnetic susceptibility and ESR results also show that there are double spin states of iron(III) ions within the crystal lattice of both compounds.     

Spectroscopic characterization of a high-potential monohaem cytochrome from Wolinella succinogenes, a nitrate-respiring organism. Redox and spin equilibria studies

When purified, a high-potential c-type monohaem cytochrome from the nitrate-respiring organism, Wollinella succinogenes (VPI 10659), displayed a minimum molecular mass of 8.2 kDa and 0.9 mol iron and 0.95 mol haem groups/mol protein. Visible light spectroscopy suggested the presence of an equilibrium between two ligand arrangements around the haem, i.e. an absorption band at 695 nm characteristic of haem-methionine coordination (low-spin form) coexisting with a high-spin form revealed by a band at 619 nm and a shoulder at 498 nm. The mid-point redox potential measured by visible redox titration of the low-spin form was approximately +100 mV. Binding cyanide (Ka = 5 x 10(5) M-1) resulted in the displacement of the methionyl axial residue, and full conversion to a low-spin, cyanide-bound form. Structural features were studied by 300-MHz 1H-NMR spectroscopy. In the oxidized state, the pH dependence of the haem methyl resonances (pH range 5-10) and the magnetic susceptibility measurements (using an NMR method) were consistent with the visible light spectroscopic data for the presence of a high-spin/low-spin equilibrium with a transition pKa of 7.3. The spin equilibrium was fast on the NMR time scale. The haem methyl resonances presented large downfield chemical shifts. An unusually broad methyl resonance at around 35 ppm (pH = 7.5, 25 degrees C) was extremely temperature-dependent [delta(323 K) - delta(273 K) = 7.2 ppm] and was assigned to the S-CH3 group of the axial methionine. In the ferrous state only a low-spin form is present. The haem meso protons, the methyl group and the methylene protons from the axial methionine were identified in the reduced form. The resonances from the aromatic residues (three tyrosines and one phenylalanine) were also assigned. Detailed monitoring of the NMR-redox pattern of the monohaem cytochrome from the fully reduced up to the fully oxidized state revealed that the rate of the intermolecular electronic exchange process was approximately 6 x 10(6) M-1 s-1 at 303 K and pH = 6.31. A dihaem cytochrome also present in the crude cell extract and purified to a homogeneous state, exhibited a molecular mass of 11 kDa and contained 2.43 mol iron and 1.89 mol haem c moieties/mol cytochrome. The absorption spectrum in the visible region exhibited no band at 695 nm, suggesting that methione is not a ligand for either of the two haems. Recovery of only small amounts of this protein prevented more detailed structural analyzes.     

Variable-temperature magnetic-circular-dichroism spectra of cytochrome c oxidase and its derivatives

A detailed study of the effect of temperature on the m.c.d. (magnetic circular dichroism) spectra of cytochrome c oxidase and some of its derivatives was undertaken to characterize the spin states of haem a and a(3). The fully reduced enzyme contains haem a(3) (2+) in its high-spin form and haem a(2+) in the low-spin state. This conclusion is reached by comparing the spectrum with that of the mixed-valence CO derivatives and its photolysis product. The cyanide derivative of the fully reduced enzyme contains both haem a and a(3) in the low-spin ferrous form. The m.c.d. spectra of the fully oxidized derivatives are consistent with the presence of one low-spin ferric haem group, assigned to a, which remains unaltered in the presence of ligands. Haem a(3) is high spin in the resting enzyme and the fluoride derivatives, and low spin in the cyanide form. The partially reduced formate and cyanide derivatives have temperature-dependent m.c.d. spectra due to the presence of high- and low-spin haem a(3) (3+) respectively. Haem a is low-spin ferrous in both. A comparison of the magnitude of the temperature-dependence of haem a(3) (3+) in the fully oxidized and partially reduced forms shows a marked difference which is tentatively ascribed to the presence of anti-ferromagnetic coupling in the fully oxidized form of the enzyme, and to its absence from the partially reduced derivatives, owing to the reduction of both Cu(2+) ions.     

Spectroscopic comparison of the heme active sites in WT KatG and its S315T mutant

KatG, the catalase-peroxidase from Mycobacterium tuberculosis, has been characterized by resonance Raman, electron spin resonance, and visible spectroscopies. The mutant KatG(S315T), which is found in about 50% of isoniazid-resistant clinical isolates, is also spectroscopically characterized. The electron spin resonance spectrum of ferrous nitrosyl KatG is consistent with a proximal histidine ligand. The Fe-His stretching vibration observed at 244 cm(-1) for ferrous wild-type KatG and KatG(S315T) confirms the imidazolate character of the proximal histidine in their five-coordinate high-spin complexes. The ferrous forms of wild-type KatG and KatG(S315T) are mixtures of six-coordinate low-spin and five-coordinate high-spin hemes. The optical and resonance Raman signatures of ferric wild-type KatG indicate that a majority of the heme exists in a five-coordinate high-spin state, but six-coordinate hemes are also present. At room temperature, more six-coordinate low-spin heme is observed in ferrous and ferric KatG(S315T) than in the WT enzyme. While the nature of the sixth ligand of LS ferric wild-type KatG is not completely clear, visible, resonance Raman, and electron spin resonance data of KatG(S315T) indicate that its sixth ligand is a neutral nitrogen donor. Possible effects of these differences on enzyme activity are discussed.