Spin state equilibria in soybean ferric leghemoglobin and its complexes with fomate and acetate

The interactions of fluoride, acetate and formate with soybean ferric leghemoglobin $\text{a}$ have been investigated by ¹H NMR spectroscopy. In the presence of fluoride or acetate leghemoglobin is locked into a high spin ferric conformation whilst the formate complex exists as an equilibrium mixture of high and low spin states. Both formate and acetate ligate directly to the iron and the different magnetic properties of the complexes are attributed to steric constraints within the heme pocket.     

Leghemoglobin. An electron paramagnetic resonance and optical spectral study of the free protein and its complexes with nicotinate and acetate.

Electron paramagnetic resonance (EPR) and optical spectra are used as probes of the heme and its ligands in ferric and ferrous leghemoglobin. The proximal ligand to the heme iron atom of ferric soybean leghemoglobin is identified as imidazole by comparison of the EPR of leghemoglobin hydroxide, azide, and cyanide with the corresponding derivatives of human hemoglobin. Optical spectra show that ferric soybean leghemoglobin near room temperature is almost entirely in the high spin state. At 77 K the optical spectrum is that of a low spin compound, while at 1.6 K the EPR is that of a low spin form resembling bis-imidazole heme. Acetate binds to ferric leghemoglobin to form a high spin complex as judged from the optical spectrum. The EPR of this complex is that of high spin ferric heme in a nearly axial environment. The complexes of ferrous leghemoglobin with substituted pyridines exhibit optical absorption maxima near 685 nm, whose absorption maxima and extinctions are strongly dependent on the nature of the substitutents of the pyridine ring; electron withdrawing groups on the pyridine ring shift the absorption maxima to lower energy. A crystal field analysis of the EPR of nicotinate derivatives of ferric leghemoblobin demonstrates that the pyridine nitrogen is also bound to the heme iron in the ferric state. These findings lead us to picture leghemoglobin as a somewhat flexible molecule in which the transition region between the E and F helices may act as a hinge, opening a small amount at higher temperature to a stable configuration in which the protein is high spin and can accommodate exogenous ligand molecules and closing at low temperature to a second stable configuration in which the protein is low spin and in which close approach of the E helix permits the distal histidine to become the principal sixth ligand.     

Unusual spin-state transitions in the reduction of ferrylmyoglobin at low temperature

Ferrylmyoglobin is reduced in γ-irradiated aqueous ethylene glycol solutions at 77 K to form a novel ferric low-spin type compound. The transition of the low-temperature product to the final high-spin ferrimyoglobin at higher temperature involves several intermediate species appearing in a sequence as temperature increases. With manifestation of the transient low-spin forms and probably the mid-spin $\text{S}\text{=}\text{3}\text{2}$ state, the process of ferrylmyoglobin reduction is found to display the spin-state transitions scarcely observed in ferric heme reactions.     

Characterization of the coral allene oxide synthase active site with UV-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy: evidence for tyrosinate ligation to the ferric enzyme heme iron

Coral allene oxide synthase (AOS), a hemoprotein with weak sequence homology to catalase, is the N-terminal domain of a naturally occurring fusion protein with an 8R-lipoxygenase. AOS converts 8R-hydroperoxyeicosatetraenoic acid to the corresponding allene oxide. The UV--visible absorption and magnetic circular dichroism spectra of ferric AOS and of its cyanide and azide complexes, and the electron paramagnetic resonance spectra of native AOS (high-spin, g = 6.56, 5.22, 2.00) and of its cyanide adduct (low-spin, g = 2.86, 2.24, 1.60) closely resemble the corresponding spectra of bovine liver catalase (BLC). These results provide strong evidence for tyrosinate ligation to the heme iron of AOS as has been established for catalases. On the other hand, the positive circular dichroism bands in the Soret region for all three derivatives of ferric AOS are almost the mirror image of those in catalase. In addition, the cyanide affinity of native AOS (K(d) = 10 mM at pH 7) is about 3 orders of magnitude lower than that of BLC. Thus, while these results conclusively support a common tyrosinate-ligated heme in AOS as in catalase, significant differences exist in the interaction between their respective heme prosthetic groups and protein environments, and in the access of small molecules to the heme iron.     

The interaction between the heme c and heme d moieties of Pseudomonas nitrite reductase as revealed by magnetic and natural circular dichroism studies.

A possibility of a heme-heme interaction between the heme $\text{c}$ and heme $\text{d}$ moieties in $\text{Pseudomonas}$ nitrite reductase was examined by using magnetic and natural circular dichroism. The MCD of the heme $\text{c}$ moiety in the ferric enzyme was similar to that of mammalian ferricytochrome $\text{c}$ in shape and intensity, whereas in the reduced state the MCD intensity was considerably smaller than that of ferrocytochrome $\text{c}$. When the heme $\text{d}$ moiety was perturbed by the complex formation with CO, imidazole or cyanide as well as by pH changes, the depressed MCD was restored to the MCD level of mammalian ferrocytochrome $\text{c}$, accompanying conformational changes around the prosthetic groups. Thus, it was concluded that the heme-heme interaction exists only in the reduced enzyme and that this interaction is released under appropriate conditions.     

Studies on the heme environment of horse heart ferric cytochrome c. Azide and imidazole complexes of ferric cytochrome c.

Horse heart ferric cytochrome c was investigated by the following three methods: (I) Light absorption spectrophotometry at 23 degrees C and 77 degrees K; (II) Electron paramagnetic resonance (EPR) spectroscopy at 20 degrees K; (III) Precise equilibrium measurements of ferric cytochrome c with azide and imidazole between 14.43 and 30.90 degrees C. I and II have demonstrated that: (1) Ferric cytochrome c azide and imidazole complexes were in the purely low spin state between 20 degrees K and 23 degrees C; (2) The energy for the three t2g orbitals calculated in one hole formalism shows that azide or imidazole bind to the heme iron in a similar manner to met-hemoglobin azide or imidazole complexes, respectively. III has demonstrated that: (1) The change of standard enthalpy and that of standard entropy were -2.3 kcal/mol and -1.6 cal/mol per degree for the azide complex formation, and -1.4 kcal/mol and 2.9 cal/mol per degree for the imidazole complex formation. (2) A linear relationship between the change of entropy and that of enthalpy was observed for the above data for the cyanide complex formation. The complex formation of ferric cytochrome c was discussed based on the results of X-ray crystallographic studies compared with hemoglobin and myoglobin.     

Spin-state equilibrium in the model complexes of azide hemoprotein

Addition of NaN3 to ferric protohemin biscoordinated with 1-methylimidazole (1-MeIm) or 2-methylimidazole (2-MeIm) in (CH3)2SO resulted in sizeable visible absorption changes, corresponding to the formation of the mixed ligand complexes, hemin X N-3 X 1-MeIm and hemin X N-3 X 2-MeIm. The visible absorption spectrum of the 1-MeIm complex was closely similar to those of azide hemoproteins, while the 2-MeIm derivative exhibited intensified 500 and 625 nm bands and depressed 540 and 570 nm peaks. The iron-bound N-3 of the model complexes exhibited two infrared stretching bands, which were assigned to the high- and low-spin peaks. The intensity of the high-spin infrared peaks increased at higher temperature. From the analyses of the infrared spectral changes, the thermodynamic values of the thermal spin equilibria were determined to be delta H = -3920 cal/mol and delta S = -11.1 e.u. for hemin X N-3 X 1-MeIm and delta H = -2150 cal/mol and delta S = 7.9 e.u. for hemin X N-3 X 2-MeIm. The thermodynamic values of the 1-MeIm complex are similar to the reported values for azide metmyoglobin, suggesting that the contribution from the nonbonded porphyrin-globin contacts to the spin equilibrium is small in azide metmyoglobin. Comparison of the delta H and delta S values among model systems indicates that delta H and delta S compensation similar to that observed in hemoprotein also holds in the models. This may suggest an underlying common denominator for the spin-equilibrium mechanisms in hemins and hemoproteins.     

Optical and magnetic resonance studies of formate binding to horse liver catalase and sperm whale myoglobin.

The binding of formate ion, a substrate for the peroxidatic reaction of catalase, has been investigated by magnetic resonance techniques. Comparative studies of formate binding to ferric myoglobin have also been performed. The nuclear magnetic relaxation (NMR) rate of formate and water protons is enhanced by the presence of ferric horse liver catalase. The enhancement is not changed significantly by the addition of cyanide, indicating that water and formate are still bound in the presence of cyanide. Formate proton to heme iron distances determined by magnetic resonance techniques indicate that formate does not directly bind to the heme iron of catalase or myoglobin but to the globin, and NMR relaxation occurs as a result of outersphere mechanisms. Evidence that water forms an innersphere complex with the iron atom of the catalase heme is presented. In similar experiments with ferric myoglobin, the addition of cyanide caused a large decrease in the enhancement of the proton relaxation rate of both formate and water, indicating the displacement of water and formate from the heme and the vicinity of the heme, respectively. Broad, high-spin, ferric ion electron paramagnetic resonance absorptions of catalase and myoglobin at room temperature obtained in the presence and absence of formate show that formate does not alter appreciably the heme environment of catalase or myoglobin or the spin state of the heme iron. Studies on the binding of formate to catalase as monitored by changes in the heme absorption spectrum in the visible region show one-to-one stoichiometry with heme concentration. However, the small changes observed in the visible region of the optical spectrum on addition of formate ion are attributed to a secondary effect of formate on the heme environment, rather than direct binding of formate to the heme moiety.     

Heme occupancy of catalase in hemoglobin-free perfused rat liver and of isolated rat liver catalase

Maximal heme occupancy, the maximal proportion of total catalase heme present in the form of Compound I, is found to be 0.4 both in the enzyme isolated from rat liver and in the peroxisomal enzyme as present in the intact cells of perfused rat liver. This indicates that the ratio of second order rate constants for catalatic decomposition and for formation of Compound I, $\text{k}{}_{\mathrm{4\prime }}\text{k}{}_1$, is equal in vitro and in vivo.Catalase was isolated from rat liver, and the extinction coefficients for Compound I and for cyanide-catalase at 640 minus 660 nm were determined. The measurement of heme occupancy of catalase in hemoglobin-free perfused rat liver was made possible by wavelength scanning as well as by dual wavelength absorbance photometry. Thus, Compound I and cyanide-catalase were demonstrated in the red region and in the Soret band region.Meeting the particular needs of organ photometry, specific metabolic transitions were used to visualize specific transitions of absorbing pigments. Compound I is specifically demonstrated by its decomposition by the hydrogen donor, methanol. A measure for total catalase heme is provided by formation of cyanide-catalase. The cyanide concentrations required are well below appearance of possible interference by other cyanide-binding hemoproteins at 640–660 nm.     

Influence of quaternary structure of the globin on thermal spin equilibria in different methemoglobin derivatives.

We have measured the paramagnetic susceptibilities of sperm whale azide metmyoglobin and of carp azide, thiocyanate, and nitrite methemoglobin in the quaternary oxy (R) and deoxy (T) structures between about 300 and 90 K, using a new sensitive superconducting magnetometer. We have also measured the pressure dependence of the high- and low-spin optical absorption bands of azide metmyoglobin and of carp azide methemoglobin in the R and T structures between 1 and 2000-4000 atmospheres. At low temperatures all the derivatives show normal Curie behavior, but above 200-250 K this is reversed, so that a thermal spin equilibrium is set up and the paramagnetic susceptibilities rise steeply with rising temperature. At all temperatures the effective magnetic moments in the T structure are higher than in the R structure. The magnetic data for azide methemoglobin have been subjected to detailed analysis. Below 250 K the magnetic moment in the R structure is 1.98 microB, characteristic of pure low spin, but that in the T structure is 2.80 microB, suggestive of a random mixture of high- and low-spin centers which have become frozen in by the immobility of the surrounding protein. Comparison of the thermal spin equilibria above 250 K shows that in the T structure the equilibrium is biased toward higher spin by the equivalent of about 1 kcal/mol relative to the R structure. Hydrostatic pressure reduces the optical density of the high-spin band at 630 nm and increases that of the low-spin bands at 541 and 573 nm. We have calibrated the optical density of the band at 630 nm against the measured paramagnetic susceptibilities of sperm whale azide metmyoglobin and carp azide methemoglobin in the R and T structures and have used this calibration to determine the dependence of the spin equilibria on hydrostatic pressure; this has allowed us to calculate the volume contraction associated with the transition from the fully high to the fully low-spin state. This amounts to -6.7 and -13.3 mL/mol heme for carp azide methemoglobins in the R and T structures, respectively, and to -12.5 mL/mol heme for azide metmyoglobin. These volume contractions are larger than those of about -4 mL/mol Fe found in synthetic iron chelates. Apparently stereochemical changes of the globin surrounding the heme also contribute to the volume changes; these must be larger in the T than in the R structure. The significance of these observations for the mechanism of heme-heme interaction is discussed.     

Magnetic circular dichroism studies of bovine liver catalase.

Absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of beef liver catalase at pH 5.0 and 6.9, and its complexes with NaF, KCNO, NaCNS, NaN3 and NaCN, have been measured between 250 nm and 700 nm at room temperature. The pH 6.9 native catalase MCD shows the presence of several additional transitions not resolved in the absorption spectrum. While these bands can be seen in the spectra of all the derivatives, with the exception of the cyanide, their relative intensities changes considerably between complexes. Of special interest in the MCD of ferric hemes is the signal intensity at about 400 nm and 620 nm. The data indicate that the MCD intensity at 620 nm increases as the high spin iron porphyrin fraction increases, reaching a maximum with the fluoride complex. The 430 nm band intensity increases as the proportion of low spin iron increases, reaching a maximum with the cyanide complex. The MCD spectra also indicate clearly the existence of spin mixtures in the complexes with CNO-, CNS-, and N3-, where both the 430 nm and 620 nm bands have appreciable intensity. It is significant that despite almost identical absorption spectra the CNS- complex has higher fraction of low spin iron than either the CNO- or the N3- species. The differences between the pH 5 and 6.9 MCD spectra of the native catalase suggest that the environment of the heme centre is sensitive to protonation.     

Gamma-glutamyltransferase activity of liver plasma membrane: induction following chronic alcohol consumption

The proton nuclear magnetic resonance spectra of soybean ferric leghemoglobin $\text{a}$ in the low-spin cyanide and nicotinate complexes have been assigned by specific deuteration of heme methyl groups. The assignments differ from those obtained solely from nuclear Overhauser enhancement measurements and are indicative of a proximal histidyl imidazole-hemin interaction which is very similar to that found in sperm whale myoglobin. The absence of a hyperfine shifted exchangeable NH peak for the distal histidine in leghemoglobin suggests either a very different orientation for this distal ligand or a significantly faster exchange rate with bulk solvent than found in myoglobin.     

Iron . bleomycin . DNA system evidence of a long-lived bleomycin . iron . oxygen intermediate

When Fe(II) is added to a bleomycin. DNA mixture in the presence of air a long-lived $\text{(}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 45}\text{minutes}\text{)}$ EPR silent species (I′) is formed; the circular dichroism and absorption spectra of which have been characterized. This complex slowly decays yielding a ferric complex (III′) analogous to the well known low spin Fe(III). BLM species.     

On the purification of nitrite reductase from Thiobacillus denitrificans and its reaction with nitrite under reducing conditions.

Nitrite reductase (cytochrome $\text{cd}$) from $\text{T. denitrificans}$ has been crystallized in high yield in three simple and rapid steps. The spectral absorption ratio at 408 to 280 nm was 1.52. Light absorption spectra in the oxidized and reduced states were virtually identical to those of nitrite reductase from $\text{P. aeruginosa}$. EPR spectroscopy of nitrite reductase at 12° showed a low-spin ferric heme resonance with g-values at 2.52, 2.45 and 1.73 assigned to the d-heme. Reaction of nitrite reductase with nitrite in the presence of the reducing systems [(ascorbate + PMS) or sulfide] resulted in the formation of nitric oxide (confirmed by gas chromatography) which reacted with both $\text{c}$- and $\text{d}$-hemes of nitrite reductase yielding an EPR-detectable enzyme-NO complex with g-values at 2.07, 2.04 and 1.99 and a ¹⁴N hyperfine splitting constant of 22.5 gauss. The amount of nitric oxide produced enzymatically with sulfide as electron donor was only 5% of that found when ascorbate plus PMS served as reductant.To our knowledge the detection of the unique enzyme-NO complex is the first definitive EPR evidence for the mandatory liganding of nitric oxide with pure nitrite reductase during nitrite reduction.     

Fluorescence studies of a cytochrome C mixed phospholipid complex

The fluorescent probe 8-anilino-1-naphthalene sulphonic acid (ANS) has been used to demonstrate the accessibility of the heme of cytochrome $\text{c}$ — mixed mitochondrial phospholipid complexes to the solvent. Contrary to earlier reports fluorescence techniques using ANS do not detect redox induced conformational changes of these complexes.     

Proton and nitrogen-15 NMR spectroscopic studies of hydrogen ion-dependent pseudo-halide ion binding to chloroperoxidase

The proton nuclear magnetic resonance spectra of several chloroperoxidase-inhibitor complexes have been investigated. Titrations of chloroperoxidase with azide, thiocyanate, cyanate, or nitrite ions indicate that only the chloroperoxidase-thiocyanate complex exhibits slow ligand exchange on the 360-MHz NMR time scale. The temperature dependence of the proton NMR spectra of the complexes suggests that, although the complexes are predominantly low-spin ferric heme iron, a spin equilibrium is present presumably between S = 1/2 and S = 5/2 states. The pH dependence of the proton NMR spectra of the psuedo-halide-chloroperoxidase complexes was examined at 360 and 90 MHz. Chloroperoxidase complexes with azide and cyanate show similar behavior; 360-MHz proton spectra are readily observed at low pH (less than 5.0) but not at high pH. At high pH, the ligand exchange rate falls in an intermediate time range. When the complexes are examined at 90 MHz, however, spectra consisting of averaged signals are observed. The chloroperoxidase-thiocyanate complex does not form at high pH values; the proton NMR spectrum observed is that of native chloroperoxidase. The pKa for the chloroperoxidase-thiocyanate heme-linked ionizable amino acid residue falls between 4.2 and 5.0. Only an averaged azide signal was observed in the nitrogen-15 NMR spectra for solutions that contained the azide complex of chloroperoxidase, horseradish peroxidase, and myoglobin.     

Influence of temperature on Photosystem II electron transfer reactions

The influence of temperature on the rate of reduction of P-680⁺, the primary donor of Photosystem II, has been studied in the range 5–294 K, in chloroplasts and subchloroplasts particles. P-680 was oxidized by a short laser flash. Its oxidation state was followed by the absorption level at 820 nm, and its reduction attributed to two mechanisms: electron donation from electron donor D₁ and electron return from the primary plastoquinone (back-reaction).Between 294 and approx. 200 K, the rate of the back-reaction, on a logarithmic scale, is a linear function of the reciprocal of the absolute temperature, corresponding to an activation energy between 3.3 and 3.7 kcal · mol^?1, in all of the materials examined (chloroplasts treated at low pH or with Tris; particles prepared with digitonin). Between approx. 200 K and 5 K the rate of the back-reaction is temperature independent, with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{ms}$. In untreated chloroplasts we measured a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.7 ms for the back-reaction at 77 and 5 K.The rate of electron donation from the donor D₁ has been measured in darkadapted Tris-treated chloroplasts, in the range 294–260 K. This rate is strongly affected by temperature. An activation energy of 11 kcal · mol^?1 was determined for this reaction.In subchloroplast particles prepared with Triton X-100 the signals due to P-680 were contaminated by absorption changes due to the triplet state of chlorophyll a. This triplet state has been examined with pure chlorophyll a in Triton X-100. An Arrhenius plot of its rate of decay shows a temperature-dependent region (292–220 K) with an activation energy of 9 kcal · mol^?1, and a temperature-independent region (below 200 K) with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1}\text{ms}$.     

Quaternary structure and spin-state transition in azide methemoglobin A

The temperature-dependent ultraviolet and visible absorption changes of human azide methemoglobin with and without inositol hexaphosphate (IHP) were examined in a 4'-35 degrees C range. The 537-nm absorption change of IHP-free hemoglobin was about 1.2-fold larger than that of IHP-bound hemoglobin. The data were analyzed by considering the thermal spin equilibrium within the R and T conformers and the quaternary equilibrium between the two conformers. The spin equilibrium analysis suggested that the T conformer has a larger high-spin content than the R conformer. The quaternary equilibrium analysis, on the other hand, showed that the T conformer is more populated at lower temperature. The thermodynamic values for the quaternary equilibrium were determined to be delta H = -13.3 kcal/mol and delta S = -47.6 eu. The large negative delta H and delta S values were compensated for each other to give a small energy difference between the two quaternary states, e.g., delta G4 = 670 cal/mol of tetramer at 20 degrees C. The coincidence of the temperature-dependent IHP-induced changes in the visible and ultraviolet absorptions of heme and aromatic chromophores at the subunit boundaries suggested that the quaternary transition energy is not localized at heme moiety. The reverse temperature dependence of the T conformer fraction as compared with the high-spin fraction of heme iron was interpreted as indicating that the appearance of the T state is not directly coupled with an increase in the strain of Fe-N(F8 His) linkage in azide methemoglobin A.     

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.     

Azide binding to the cytochrome c ferric heme octapeptide. A model for anion binding to the active site of high spin ferric heme proteins.

Equilibrium constants for the binding of azide to the ferric heme c octapeptide in 50% ethylene glycol 50% buffer were measured spectrophotometrically. The equilibrium constant for azide binding at 20 degrees C and pH* 7.4 is 29.2, which is approximately 3 to 4 orders of magnitude lower than that observed for azide binding to various ferric hemeproteins. The equilibrium constant was indepent of pH* in the range from 7 to 8. Equilibrium constants at several temperatures exhibited an apparent van't Hoff relationship yielding thermodynamic values of delta H0 = -26,100 J/mol (-6240 cal/mol) and delta S0 = -61.5 J/0K mol (-14.7 e.u.). Comparison of these values to the values for the heme proteins enables one to explain the differences in equiliberium constants in terms of differences in the polarity of the heme environments. The results are consistent with the concept that the oxygen affinity of heme complexes increases with the polarity of the heme environment. The data also suggest that an increase in the polarity of the heme environment should result in a corresponding increase in the susceptibility of ferrous heme dioxygen complexes toward oxidation by the dioxygen.