Theoretical study of the distal-side steric and electrostatic effects on the vibrational characteristics of the FeCO unit of the carbonylheme proteins and their models.

The vibronic theory of activation and quantum chemical intermediate neglect of differential overlap (INDO) calculations are used to study the activation of carbon monoxide (change of the C-O bond index and force field constant) by the imidazole complex with heme in dependence on the distortion of the porphyrin ring, geometry of the CO coordination, iron-carbon and iron-imidazole distances, iron displacement out of the porphyrin plane, and presence of the charged groups in the heme environment. It is shown that the main contribution to the CO activation stems from the change in the sigma donation from the 5 sigma CO orbital to iron, and back-bonding from the iron to the 2 pi orbital of CO. It follows from the results that none of the studied distortions can explain, by itself, the wide variation of the C-O vibrational frequency in the experimentally studied model compounds and heme proteins. To study the dependence of the properties of the FeCO unit on the presence of charged groups in the heme environment, the latter are simulated by the homogeneous electric field and point charges of different magnitude and location. The results show that charged groups can strongly affect the strength of the C-O bond and its vibrational frequency. It is found that the charges located on the distal side of the heme plane can affect the Fe-C and C-O bond indexes (and, consequently, the Fe-C and C-O vibrational frequencies), both in the same and in opposite directions, depending on their position. The theoretical results allow us to understand the peculiarities of the effect of charged groups on the properties of the FeCO unit both in heme proteins and in their model compounds.     

Correct interpretation of heme protein spectra allows distinguishing between the heme and the protein dynamics

It is shown by using the vibronic approach that the iron displacement out of the porphyrin plane in deoxyheme proteins intermixes the porphyrin pi and axial iron-histidine sigma electronic subsystems. This intermixing explains the substantial coupling of the iron-histidine vibration to the heme Soret excitation, the appearance of the iron-histidine band in the corresponding resonance Raman spectra, and a number of other experimental data, including the dependence of the iron-histidine vibrational frequency on the extent of the iron displacement out of the porphyrin plane. This dependence implies that there is an anharmonic coupling between the corresponding vibrations, which is shown to be the cause of the specific temperature dependence of the iron-histidine band. The anharmonic coupling and the dependence of the dipole transition moment of the charge transfer optical absorption band III on the iron-porphyrin distance cause the anomalous temperature and pressure dependencies of this band. It is shown that the change in both the magnitude and the distribution of the iron-porphyrin distance is expected to affect the band III intensity. Consequently, the stationarity of the band III intensity can be considered as a signature of the stationarity of the iron-porphyrin distance and its distribution in deoxyheme proteins, whereas the band III position and width could be also affected by the change in the protein electric field, caused by the protein globule dynamics.     

M?ssbauer spectroscopic study of compound ES of cytochrome c peroxidase.

M?ssbauer spectra of Compound ES of cytochrome c peroxidase have been observed over a range of temperature and applied magnetic field. These have been interpreted in terms of a crystal field model of the iron site in which the iron is assumed to be in the Fe(IV) state with unpaired spin S = 1. Detailed least-squares fitting of the spectra fitting of the spectra indicates that both the electric field gradient choice of a single parameter, the axial crystal field, the magnetic properties are well reproduced. The model also provides the observed positive sign for the electric field gradient interaction, but overestimates its magnitude. This apparent discrepnancy may be caused by the presence of significant electronic charge in filled bonding orbitals, a feature which is in keeping with expected covalent charge compensation of the extreme oxidation state. There is no evidence in the M?ssbauer spectra of interaction between the iron and the ESR-visible free radical. This suggests they are well separated.     

Liposomal heme as oxygen carrier under semi-physiological conditions. Orientation study of heme embedded in a phospholipid bilayer by an electrooptical method

The meso-tetra(alpha,alpha,alpha,alpha(o-pivalamidophenyl]porphinato iron-mono(1-lauryl-2-methylimidazole) complex embedded in the bilayer of dimyristoylphosphatidylcholine (liposomal heme) binds molecular oxygen reversibly at pH 7 and 37 degrees C. Orientation of the iron porphyrin complex in the phospholipid bilayer was studied by electric birefringence and dichroism. It was observed that both the phospholipid bibilayer of liposome and the porphyrin plane are oriented nearly in parallel to the electric field. Therefore the angle between the porphyrin plane and the bilayer is considered to be practically small.     

Imidazole, the ligand trans to mercaptide in ferric cytochrome P-450. An EPR study of proteins and model compounds.

A crystal field analysis of EPR data for various low spin ferric cytochromes P-450 suggests that in all of them, regardless of source or method of induction, the heme ligands are a sulfur atom, presumably from cysteine, and an imidazole from histidine. The imidazole can be displaced in the ferric protein by cyanide, guanidine, or by an amine, analogous to its displacement by CO or NO in the ferrous protein. The resulting changes in the EPR parameters for the ferric protein are consistent with similar substitutions in heme thiol model compounds. The analysis of the latter can be understood on the basis of alterations of the electronic structure of the ligands to the heme iron.     

M?ssbauer investigations of high-spin ferrous heme proteins. I. Cytochrome P-450.

Anaerobically reduced samples of cytochrome P-450 from Pseudomonas putida were studied by M?ssbauer spectroscopy. In the presence of an applied magnetic field the high-spin ferrous heme iron showed an intricate pattern of electric and magnetic hyperfine interactions which could be parametrized successfully in terms of a spin Hamiltonian formalism. The results imply a very low (triclinic) symmetry of the heme iron. The effects of the ligand environment and of spin-orbit coupling result in a large zero-field splitting of the electronic ground state. The electronic ground state. The electric-field gradient tensor is characterized by a large asymmetry parameter, and its principal axes are rotated substantially from the frame that defines the zero-field splitting. This study shows that high-field M?ssbauer spectroscopy provides a unique tool for structural investigations of high-spin ferrous compounds and can substitute, under suitable conditions, for magnetic susceptibility measurements. The present paper focuses on the methodology and data analysis; in the subsequent paper the data obtained for P-450 are compared with new results obtained for hemoglobin, chloroperoxidase, and horseradish peroxidase.     

Dynamic structural disorder of the FeO<Subscript>2</Subscript> moiety in oxymyoglobin studied by nuclear resonant forward scattering of synchrotron radiation

Oxy- as well as deoxymyoglobin exhibit a pronounced temperature dependence of the quadrupole splitting of the heme iron as detected by conventional M?ssbauer spectroscopy. With nuclear resonant forward scattering (NFS) of synchrotron radiation, which can be viewed as M?ssbauer spectroscopy in the time domain, it is shown that this spectroscopic behavior, although it is phenomenologically similar in the two cases, is based on completely different physical mechanisms. It is demonstrated that stochastic fluctuations of the iron electric field gradient in MbO(2), which are due to the dynamic structural disorder of the FeO(2) moiety, are the reason for the temperature-dependent alterations of the coherent quantum beat pattern in the NFS spectra of MbO(2), in contrast to deoxyMb where transitions between orbital states of iron take place. This subtle spectroscopic difference cannot be inferred from conventional M?ssbauer spectroscopy.     

A single mutation in cytochrome P450 BM3 induces the conformational rearrangement seen upon substrate binding in the wild-type enzyme

The multidomain fatty-acid hydroxylase flavocytochrome P450 BM3 has been studied as a paradigm model for eukaryotic microsomal P450 enzymes because of its homology to eukaryotic family 4 P450 enzymes and its use of a eukaryotic-like diflavin reductase redox partner. High-resolution crystal structures have led to the proposal that substrate-induced conformational changes lead to removal of water as the sixth ligand to the heme iron. Concomitant changes in the heme iron spin state and heme iron reduction potential help to trigger electron transfer from the reductase and to initiate catalysis. Surprisingly, the crystal structure of the substrate-free A264E heme domain mutant reveals the enzyme to be in the conformation observed for substrate-bound wild-type P450, but with the iron in the low-spin state. This provides strong evidence that the spin-state shift observed upon substrate binding in wild-type P450 BM3 not only is caused indirectly by structural changes in the protein, but is a direct consequence of the presence of the substrate itself, similar to what has been observed for P450cam. The crystal structure of the palmitoleate-bound A264E mutant reveals that substrate binding promotes heme ligation by Glu(264), with little other difference from the palmitoleate-bound wild-type structure observable. Despite having a protein-derived sixth heme ligand in the substrate-bound form, the A264E mutant is catalytically active, providing further indication for structural rearrangement of the active site upon reduction of the heme iron, including displacement of the glutamate ligand to allow binding of dioxygen.     

Liposomal heme as oxygen carrier under semi-physiological conditions orientation study of heme embedded in a phospholipid bilayer by an electrooptical method

The meso-tetra(α,α,α,α(o-pivalamidophenyl))porphinato iron-mono(1-lauryl-2-methylimidazole) complex embedded in the bilayer of dimyristoylphosphatidylcholine (liposomal heme) binds molecular oxygen reversibly at pH 7 and 37°C. Orientation of the iron porphyrin complex in the phospholipid bilayer was studied by electric birefringence and dichroism. It was observed that both the phospholipid bibilayer of liposome and the porphyrin plane are oriented nearly in parallel to the electric field. Therefore the angle between the porphyrin plane and the bilayer is considered to be practically small.     

The structure of ferrocytochrome b5 at 2.8 A resolution.

Crystals of cytochrome b5 reduced by sodium dithionite are isomorphous with the oxidized form. An electron density difference map between the two forms was calculated at 2.8 A resolution. There are no changes in main chain conformation or internal side chain orientation upon reduction. However, an ion becomes attached at the entrance of the heme crevice causing displacement of a surface lysine side chain on an adjacent molecule. The ion, identified as a cation by the nature of its coordinating ligands, appears to neutralize one of the heme propionate groups which is partially buried. It is proposed that the negatively charged propionate serves to neutralize the net formal positive charge on the heme iron in the oxidized cytochrome and that the neutralization of the heme iron upon reduction then leads to binding of a cation to the propionate.     

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.     

Mössbauer Effect in Hemoglobin and Some Iron-Containing Biological Compounds

The M?ssbauer effect in Fe(57) has been used to study the molecules, hemoglobin, O(2)-hemoglobin, CO(2)-hemoglobin, and CO-hemoglobin (within red cells) and the molecules, hemin and hematin (in the crystalline state). Quadrupole splittings and isomeric shifts observed in the M?ssbauer spectra of these molecules are tabulated. The temperature dependence of the quadrupole splitting and relative recoil-free fraction for hemoglobin with different ligands has been investigated. An estimate of the Debye-Waller factor in O(2)-hemoglobin at 5 degrees K is 0.83. An asymmetry in the quadrupole splitting observed in hemoglobin is attributed to a directional dependence of the recoil-free fraction which establishes the sign of the electric field gradient in the molecule and indicates that the lowest lying d orbital of the Fe atoms is |xy>. This asymmetry indicates that the iron atoms in hemoglobin are vibrating farther perpendicular to the heme planes than parallel to them, and, in fact, the ratio of the mean square displacements perpendicular and parallel to the heme planes in hemoglobin is approximately 5.5 at 5 degrees K. The temperature dependence of the quadrupole splitting in hemoglobin has been used to estimate a splitting between the lowest lying iron atom d orbitals of approximately 420 cm(-1).     

Iron normal mode dynamics in (nitrosyl)iron(II)tetraphenylporphyrin from X-ray nuclear resonance data

The complete iron atom vibrational spectrum has been obtained by refinement of normal mode calculations to nuclear inelastic x-ray absorption data from (nitrosyl)iron(II)tetraphenylporphyrin, FeTPP(NO), a useful model for heme dynamics in myoglobin and other heme proteins. Nuclear resonance vibrational spectroscopy (NRVS) provides a direct measurement of the frequency and iron amplitude for all normal modes involving significant displacement of (57)Fe. The NRVS measurements on isotopically enriched single crystals permit determination of heme in-plane and out-of-plane modes. Excellent agreement between the calculated and experimental values of frequency and iron amplitude for each mode is achieved by a force-field refinement. Significantly, we find that the presence of the phenyl groups and the NO ligand leads to substantial mixing of the porphyrin core modes. This first picture of the entire iron vibrational density of states for a porphyrin compound provides an improved model for the role of iron atom dynamics in the biological functioning of heme proteins.     

Resonance Raman investigation of the effects of copper binding to iron-mesoporphyrin.histidine-rich glycoprotein complexes.

Histidine-rich glycoprotein (HRG) binds both hemes and metal ions simultaneously with evidence for interaction between the two. This study uses resonance Raman and optical absorption spectroscopies to examine the heme environment of the 1:1 iron-mesoporphyrin.HRG complex in its oxidized, reduced and CO-bound forms in the absence and presence of copper. Significant perturbation of Fe(3+)-mesoporphyrin.HRG is induced by Cu2+ binding to the protein. Specifically, high frequency heme resonance Raman bands indicative of low-spin, six-coordinate iron before Cu2+ binding exhibit monotonic intensity shifts to bands representing high-spin, five-coordinate iron. The latter coordination is in contrast to that found in hemoglobin and myoglobin, and explains the Cu(2+)-induced decrease and broadening of the Fe(3+)-mesoporphyrin.HRG Soret band concomitant with the increase in the high-spin marker band at 620 nm. After dithionite reduction, the Fe(2+)-mesoporphyrin.HRG complex displays high frequency resonance Raman bands characteristic of low-spin heme and no iron-histidine stretch, which together suggest six-coordinate iron. Furthermore, the local heme environment of the complex is not altered by the binding of Cu1+. CO-bound Fe(2+)-mesoporphyrin.HRG exhibits bands in the high and low frequency regions similar to those of other CO-bound heme proteins except that the iron-CO stretch at 505 cm-1 is unusually broad with delta nu approximately 30 cm-1. The dynamics of CO photolysis and rebinding to Fe(2+)-mesoporphyrin.HRG are also distinctive. The net quantum yield for photolysis at 10 ns is low relative to most heme proteins, which may be attributed to very rapid geminate recombination. A similar low net quantum yield and broad iron-CO stretch have so far only been observed in a dimeric cytochrome c' from Chromatium vinosum. Furthermore, the photolytic transient of Fe(2+)-mesoporphyrin.HRG lacks bands corresponding to high-spin, five-coordinate iron as is found in hemoglobin and myoglobin under similar experimental conditions, suggesting iron hexacoordination before CO recombination. These data are consistent with a closely packed distal heme pocket that hinders ligand diffusion into the surrounding solvent.     

An EPR analysis of cyanide-resistant mitochondria isolated from the mutant poky strain of Neurospora crassa.

An analysis of the paramagnetic components present in mitochondria isolated from the poky mutant of Neurospora crassa is described. The study was undertaken with a view to shedding light on the nature of the cyanide- and antimycin A-resistant alternative terminal oxidase which is present in these preparations. Of the ferredoxin-type iron-sulfure centers, only Centers S-1 and S-2 of succinate dehydrogenase could be detected in significant quantities. Paramagnetic centers attributable to Site I were virtually absent. In the oxidized state, at least two 'high potential iron sulfur' centers could be distinguished and these were attributed to Center S-3 of succinate dehydrogenase and a second component analogous to that found in mammalian systems. Much of the Center S-3 signal was in a highly distorted state which was apparently dependent upon the presence of an accompanying free radical species. At lower field positions, a succinate-reducible signal peaking around g = 3.15 was found. This signal is caused by a low spin heme species, presumably the cytochrome c which is the only major cytochrome in these mitochondria. At even lower field positions, signals attributable to iron in a field of low symmetry at g = 4.3 and multiple high spin heme species around g = 6, could be distinguished. The effects of salicylhydroxamic acid, an inhibitor of the alternative oxidase, were tested on these components. Effects could be seen on at least one high spin heme component and also partially upon the distorted Center S-3 signal converting part of it to a signal indistinguishable from center S-3. Some increase in the g = 4.3 iron signal was also noted. No effects of the inhibitor on the ferredoxin-type centers were detected.     

X-ray structure and refinement of carbon-monoxy (Fe II)-myoglobin at 1.5 A resolution

The structure of carbon-monoxy (Fe II) myoglobin at 260 K has been solved at a resolution of 1.5 A by X-ray diffraction and a model refined against the X-ray data by restrained least-squares. The CO ligand is disordered and distorted from the linear conformation seen in model compounds. At least two conformations, with Fe--C--O angles of 140 degrees and 120 degrees, are required to model the system. The heme pocket is significantly larger than in deoxy-myoglobin because the distal residues have relaxed around the ligand; the largest displacement occurs for the distal histidine side-chain, which moves more than 1.4 A on ligand binding. The side-chain of Arg45 (CD3) is disordered and apparently exists in two equally populated conformations. One of these does not block the motion of the distal histidine out of the binding pocket, suggesting a mechanism for ligand entry. The heme group is planar (root-mean-square deviation from planarity is 0.08 A) with no doming of the pyrrole groups. The Fe--N epsilon 2 (His93) bond length is 2.2 A and the Fe--C bond length in the CO complex is 1.9 A. The iron is the least-squares plane of the heme, and this leads to the proximal histidine moving by 0.4 A relative to its position in deoxy-myoglobin. This shift correlates with a global structural change, with the proximal part of the molecule translated towards the heme plane.     

The effect of cytochrome P-450cam on the NMR relaxation rate of water protons.

Cytochrome P-450cam in the native, substrate-free state (Fe3+, S = 1/2) substantially reduces the NMR relaxation times, T1 and T2, of water protons. Temperature and frequency dependences of T1 and T2 were measured; they are consistent with a model of one or two protons exchanging between a binding site on a heme ligand and bulk water. The relevant parameters of this model have been deduced from the data. The spin relaxation time of the heme iron, tau S similar to 0.5 ns at 25 degrees C, is unusually long for a low spin ferric heme protein but is compatible with the line widths measured for paramagnetically shifted heme resonances. The proton residence time on the ligand, tau M similar to 1 microsecond at 25 degrees C, follows an Arrhenius law with activation energy EM similar to 15 kcal/mol. A scalar hyperfine interaction A/h = 2.2 MHz (3.1 MHz for one-proton exchange) of the found proton(s) with the heme iron is deduced from the difference between T1 and T2 observed in the fast exchange limit. The iron-proton distance is found to be 2.9 A (2.6 A for one-proton exchange). Variation of pH between pH 6.4 and 8.6 does not affect T1. The bearing of these results on the question of the axial heme ligand is discussed.     

A new spatial structure for the axial methionine observed in cytochrome c5 from Pseudomonas mendocina. Correlations with the electronic structure of heme c

Cytochrome c5 from Pseudomonas mendocina has been isolated and the coordination geometry at the heme iron was investigated by 1H nuclear magnetic resonance and circular dichroism spectroscopy. Individual assignments were obtained for heme c and the axial ligands. From studies of nuclear Overhauser enhancements the axial histidine imidazole ring orientation relative to the heme group was found to coincide with that of other c-type cytochromes. In contrast, a new structure was observed for the axial methionine. This includes S chirality at the iron-bound sulfur atom, but compared to cytochromes c-551 from Pseudomonads and Rhodopseudomonas gelatinosa, which also contain S-chiral methionine, the spatial arrangement of the gamma- and beta-methylene groups and the alpha carbon of methionine is markedly different. Analysis of the electron spin density distribution in ferricytochrome c5 in the light of this new coordination geometry provides additional support for the hypothesis that the electronic structure of heme c is primarily governed by the orientation of the sp3 lone-pair orbital of the axial sulfur atom with respect to the heme plane.     

Probing the local electronic and geometric properties of the heme iron center in a H-NOX domain

Heme-Nitric oxide and/or OXygen binding (H-NOX) proteins are a family of diatomic gas binding hemoproteins that have attracted intense research interest. Here we employ X-ray absorption near-edge structure (XANES) spectroscopy to study the nitric oxide (NO) binding site of H-NOX. This is the first time this technique has been utilized to examine the NO/H-NOX signaling pathway. XANES spectra of wildtype and a point mutant (proline 115 to alanine, P115A) of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) were obtained and analyzed for ferrous and ferric complexes of the protein. This work provides specific structural characterization of the solution state of several Tt H-NOX ferrous complexes (− unligated, − NO, and − CO) that were previously unavailable. Our iron K-edges indicate effective charge on the iron center in the various complexes and report on the electronic environment of heme iron. We analyzed the ligand field indicator ratio (LFIR), which is extracted from XANES spectra, for each complex, providing an understanding of ligand field strength, spin state of the central iron, movement of the iron atom upon ligation, and ligand binding properties. In particular, our LFIRs indicate that the heme iron is dramatically displaced towards the distal pocket during ligand binding. Based on these results, we propose that iron displacement towards the distal heme pocket is an essential step in signal initiation in H-NOX proteins. This provides a mechanistic link between ligand binding and the changes in heme and protein conformation that have been observed for H-NOX family members during signaling.     

Mössbauer spectroscopic study of compound es of cytochrome c peroxidase

Mössbauer spectra of Compound ES of cytochrome c peroxidase have been observed over a range of temperature and applied magnetic field. These have been interpreted in terms of a crystal field model of the iron site in which the iron is assumed to be in the Fe(IV) state with unpaired spin S = 1. Detailed least-squares fitting of the spectra indicates that both the electric field gradient and the magnetic hyperfine interactions exhibit axial symmetry. With the choice of a single parameter, the axial crystal field, the magnetic properties are well reproduced. The model also provides the observed positive sign for the electric field gradient interaction, but overestimates its magnitude. This apparent discrepancy may be caused by the presence of significant electronic charge in filled bonding orbitals, a feature which is in keeping with expected covalent charge compensation of the extreme oxidation state. There is no evidence in the Mössbauer spectra of interaction between the iron and ESR-visible free radical. The suggests they are well separated.