Heme methyl 1H chemical shifts as structural parameters in some low-spin ferriheme proteins

 The different paramagnetic shifts of the four methyl groups in ferriheme proteins have been described as being due to the effect of the axial ligand nodal plane orientation. An equation, heuristically found and theoretically explained, describing the relation between contact and pseudocontact shifts and the position of the axial ligand(s) has been derived for bis-histidine ferriheme proteins and for cyanide-histidine ferriheme proteins. The values of the heuristic parameters contained in the equations were found by fitting the shifts of bovine cytochrome b ₅ and several bis-histidine cytochromes c ₃ and histidine-cyanide systems. The agreement between the observed and the calculated shifts was found to be good. Therefore, by taking advantage of this study, information on the position of the axial ligands, that can be used as a constraint for structure determination, can be obtained from the shifts of the methyl protons. Received: 13 April 1999 / Accepted: 4 June 1999     

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.     

Orientation of the axial ligands and magnetic properties of the hemes in the triheme ferricytochrome PpcA from G. sulfurreducens determined by paramagnetic NMR

The geometry of the axial ligands of the hemes in the triheme cytochrome PpcA from Geobacter sulfurreducens was determined in solution for the ferric form using the unambiguous assignment of the NMR signals of the α-substituents of the hemes. The paramagnetic ¹³C shifts of the hemes can be used to define the heme electronic structure, the geometry of the axial ligands, and the magnetic susceptibility tensor. The latter establishes the magnitude and geometrical dependence of the pseudocontact shifts, which are crucial to warrant reliable structural constraints for a detailed structural characterization of this paramagnetic protein in solution.     

1H NMR studies of the effect of mutation at Valine45 on heme microenvironment of cytochrome b5

1D and 2D (1)H NMR were employed to probe the effects on the heme microenvironment of cytochrome b(5) caused by the mutation from Val45 to Tyr45, His45 and Glu45. Compared with wild type (WT) cytochrome b(5), in all mutants the heme ring are CCW rotated relative to the imidazole planes of axial ligands and the angles beta between two axial ligand imidazole planes are not changed, being in agreement with the temperature dependence of the shifts of the heme protons. The ratios of heme isomers (major to minor) are smaller than that in WT. The 4-vinyl group of the heme in V45Y assumes cis-orientation, being similar to that of WT, while in V45E and V45H, both cis and trans orientation are found. The relationships between the structure and biological function of the mutants are discussed in terms of the geometry of heme and axial ligands, the hydrophobicity of heme pocket and the electrostatic potential of the heme-exposed area.     

Redox-dependent structure change and hyperfine nuclear magnetic resonance shifts in cytochrome c

Proton nuclear magnetic resonance assignments for reduced and oxidized equine cytochrome c show that many individual protons exhibit different chemical shifts in the two protein forms, reflecting diamagnetic shift effects due to structure change, and in addition contact and pseudocontact shifts that occur only in the paramagnetic oxidized form. To evaluate the chemical shift differences (delta delta) for structure change, we removed the pseudocontact shift contribution by a calculation based on knowledge of the electron spin g tensor. The g-tensor parameters were determined from the delta delta values of a large set (64) of C alpha H protons at well-defined spatial positions in the oxidized horse protein. The g-tensor calculation, when repeated using only 12 available C alpha H proton resonances for cytochrome c from tuna, proved to be remarkably stable. The largest principal value of the g tensor (gz) falls precisely along the ligand bond between the heme iron and methionine-80 sulfur, while gx and gy closely match the natural heme axes defined by the pyrrole nitrogens. The derived g tensor was then used together with spatial coordinates for the oxidized form to calculate the pseudocontact shift contribution (delta pc) to proton resonances at 400 identifiable sites throughout the protein, so that the redox-dependent chemical shift discrepancy, delta delta-delta pc, could be evaluated. Large residual changes in chemical shift define the Fermi contact shifts, which are found as expected to be limited to the immediate covalent structure of the heme and its ligands and to be asymmetrically distributed over the heme. Smaller chemical shift discrepancies point to a concerted change, involving residues 39-43 and 50-60 (bottom of the protein), and to other changes in the immediate vicinity of the heme ligands. Also, the three internal water molecules are implicated in redox sensitivity. The residues found to change are in good but not perfect agreement with prior X-ray diffraction observations of subangstrom redox-related displacements in the tuna protein. The chemical shift discrepancies observed appear in the main to reflect structure-dependent diamagnetic shifts rather than hyperfine effects due to displacements in the pseudocontact shift field. Although 51 protons in 29 different residues exhibit significant chemical shift changes, the general impression is one of small structural adjustments to redox-dependent strain rather than sizeable structural displacements or rearrangements.     

Correlation of empirical magnetic susceptibility tensors and structure in low-spin haem proteins

Experimental magnetic susceptibility tensors are reported for eight haems c with bis-His coordination. These data, obtained by fitting the dipolar shifts of backbone protons in the tetrahaem cytochromes c ₃ from Desulfovibrio vulgaris and D. gigas, are analysed together with published values for other haem proteins. The x and y axes are found to rotate in the opposite sense to the axial ligands and are also counter-rotated with respect to the frontier molecular orbitals of the haem. The magnetic z-axis is close to the normal to the haem plane in each case. The magnitudes of the magnetic anisotropies are used to derive crystal field parameters and the rhombic splitting, V, is correlated with the dihedral angle between the axial ligands. Hence, it is apparent that the axial ligands are the dominant factor in determining the variation in magnetic properties between haems, and it is confirmed that “high g _max” EPR signals are a reliable indicator of near-perpendicular ligands. These results are in full agreement with the analysis of non-Curie effects and electronic structure in the His-Met coordinated cytochromes c and c ₅₅₁. Collectively, they show that the orientations of axial ligands to the haem may be estimated from single-crystal EPR data, from ¹³C NMR shifts of the haem substituents, or from NMR dipolar shifts of the polypeptide. Received: 3 September 1999 / Accepted: 10 December 1999     

Dimethyl propionate ester heme-containing cytochrome b5: structure and stability.

A derivative of rat microsomal cytochrome b5, obtained by substitution of the native heme moiety with protoporphyrin IX dimethyl ester, has been characterized by 1H and 15N NMR spectroscopy. Besides the two usual A and B forms, which depend on the orientation of the heme in the prostethic group cavity, two other minor forms have been detected which presumably indicate different conformations of the vinyl side chains. The shifts of the heme methyls, as well as the directions of the rhombic axes of the magnetic susceptibility tensor, indicate a small difference in the orientation of the imidazole planes of the histidine axial ligands. The solution structure was determined by using 1,303 meaningful NOEs and 241 pseudocontact shifts, the latter being derived from the native reduced protein. A family of 40 energy-minimized conformers was obtained with average RMSD of 0.56+/-0.09 A and 1.04+/-0.12 A for backbone and heavy atoms, respectively, and distance and pseudocontact shift penalty functions of 0.50+/-0.07 A2 and 0.51+/-0.02 ppm2. The structure shows some changes around the cavity and in particular a movement of the 60-70 backbone segment owing to the absence of two hydrogen bonds between the Ser64 backbone NH and side-chain OH and the carboxylate oxygen of propionate-7, present in the native protein. The analysis of the NMR spectra in the presence of unfolding agents indicates that this protein is less stable than the native form. The decrease in stability may be the result of the loss of the two hydrogen bonds connecting propionate-7 to Ser64 in the native protein. The available data on the reduction potential and the electron transfer rates are discussed on the basis of the present structural data.     

Reversible oxygenation of heme bound to polyvinylpyridine and polyvinylimidazole

The interaction between heme bound to poly-4-vinylpyridine (PVP) or poly-N-vinyl-2-methylimidazole (PVMI) and molecular oxygen (O₂) was studied. In this paper, the reactions of some types of heme with O₂ in organic solvents, particularly in N,N-dimethylformamide (DMF) were discussed. The free heme not bound to an axial base was easily oxidized irreversibly to hemin in DMF with bubbled O₂. The hemochromogens complexed with pyridine, imidazole, or their polymeric derivatives such as PVP and PVMI bound O₂ to one of the axial coordination sites. The characteristic absorption band assignable to the resulting oxygenated heme was observed at 402 nm. This absorption band could be changed back to the characteristic band of the reduced hemochromogen at 418 nm by removing O₂ dissolved in the DMF solution by a vacuum or by a stream of nitrogen. Thus, the hemochromogens bound to the synthetic polymers were found to adsorb and desorb O₂ reversible in DMF. When the polymeric ligands were used, the equilibrium constants in the complexation of heme with these polymers were about 10² times as large as those of the corresponding monomeric ligands. The oxygenation rates and the capacities of O₂ of the polymeric hemochromogens were larger than those of the monomeric hemochromogens. In addition, the oxygenation rate of the polymer complex was changeable owing to the conformational change of the polymeric ligand; this rate increased about ten times under the optimal condition.     

Axial perturbations on the electronic and magnetic properties of ferric porphyrins I. Effect of axial ligand basicity in bis-substituted pyridine complexes of synthetic porphyrins

The proton NMR spectra of the bis-4-substituted pyridinates of ferric tetrapheylporphyrin and octaethylporphyrin complexes have been recorded and analyzed fort he purpose of ascertaining the influence of variable axial lignad basicity on the bonding and magnetic properties of the iron. Under the conditions of slow ligand exhange where the bis stoichiometry can be established, all complexes exist exlusively in the low-spin, $\text{S}\text{=}\text{1}\text{2}$, state. The hyperfine shifts at ?60° C for both the porphyrins and axial ligands are shown to be very sensitive to the basicity of the substituted pyridine, as measured by its pK_a. For the tetraphenylporphyrin complexes, we illustrate that the pattern of the meso-phenyl hyperfine shifts permits a quantitative separation of the contact and dipolar contributions to these shifts. This separation reveals that the shift variations with pyridine pK_a are dominated by changes in the magnetic susceptibility anisotropy (dipolar shift), which decreases markedly upon lowering the pyridine basicity; ESR data support this conclusion in the few samples investigated. However, this trend in magnetic anisotropy with ligand basicity is not valid when comparing pyridines with other ligands such as imidazoles. The important change in the contact shift reflects a decrease in porphyrin → iron π change transfer as the ligand basicity is lowered. A correlation between increase in proton NMR linewidth and magnetic anisotrophy of the iron suggests that electron spin relaxation occurs via a process which couples the same levels that control the magnetic anisotropy.     

Chemical shift-based constraints for solution structure determination of paramagnetic low-spin heme proteins with bis-His and His-CN axial ligands: the cases of oxidized cytochrome b 5 and Met80Ala cyano-cytochrome c

The chemical shifts of the methyl protons of protoporphyrin IX, which are readily assigned, are related to the structural features of the axial histidine ligands in heme proteins with bis-His or His-CN axial coordination (Bertini I, Luchinat C, Parigi G, Walker FA (1999) JBIC 4:515-519). In the present paper, a module is developed which transforms the chemical shifts into a pseudo-potential energy that is a function of the dihedral angles defining the orientation of the axial ligand planes. Minimization of this pseudo-potential energy, together with the energetic contributions provided by the other constraints, yields structures consistent with the heme methyl chemical shifts. Oxidized cytochrome b(5) from rat and the cyanide derivative of the M80A mutant of yeast cytochrome c are used for test calculations. In the case of scarcity of NOEs for the axial ligands, owing to the presence of the paramagnetic center, the above structural constraints are shown to be quite precious. The newly refined structures are deposited in the PDB.     

1H NMR study of the solution molecular and electronic structure of Escherichia coli ferricytochrome b562: evidence for S = 1/2 in equilibrium S = 5/2 spin equilibrium for intact His/Met ligation

The solution 500-MHz 1H NMR spectral parameters for ferricytochrome b562, a soluble 12-kDa electron carrier from Escherichia coli with axial His/Met coordination, are shown to be strongly influenced by protein concentration and ionic strength at low pH and 25 degrees C in a manner consistent with significant aggregation at low ionic strength. At high ionic strength a well-resolved 1H NMR spectrum reveals over 40 hyperfine-shifted resonances which arise from two isomeric species in the ratio 2:1. 2D COSY and NOESY maps at 25 degrees C for the hyperfine-shifted resonances allow the assignment of a number of axial His resonances and all heme peripheral substituent peaks. The resulting asymmetric heme contact shift patterns, together with the halving of the number of lines when reconstituting with 2-fold symmetric hemin, demonstrate the molecular basis of the solution heterogeneity to be heme orientational disorder. The strongly upfield-shifted axial Met-7 resonances, characteristic of low-spin ferricytochromes c with His/Met ligation, appear upfield only at very low temperatures. At elevated temperatures, all resonances, in particular those of the axial Met, move strongly downfield. Detailed analysis of the deviation from Curie behavior for different functional groups demonstrates the presence of a low spin in equilibrium high spin equilibrium with an intact His-Fe-Met coordination. The weaker axial field in ferricytochrome b562, relative to the purely low-spin ferricytochromes c, is attributed to a perturbed iron-Met bond. The contact shifts for a coordinated Met in the high-spin state are estimated. A link between equatorial hemin and axial ligand interactions is indicated by a differential population of the high-spin form for the two hemin orientations.     

Ligand orientation of human neuroglobin obtained from solution NMR and molecular dynamics simulation as compared with X-ray crystallography

Neuroglobin, a new member of hemoprotein family, can reversibly bind oxygen and take part in many biological processes such as enzymatic reaction, signal transduction and the mitochondria function. Different from myoglobin and hemoglobin, it has a hexacoordinated heme environment, with histidyl imidazole of proximal His⁹⁶(F8) and distal His⁶⁴(E7) directly bound to the metal ion. In the present work, solution ¹H NMR spectroscopy was employed to investigate the electronic structure of heme center of wild-type met-human neuroglobin. The resonances of heme protons and key residues in the heme pocket were assigned. Two heme orientations resulting from a 180° rotation about the α-γ-meso axis with a population ratio about 2:1 were observed. Then the ¹H NMR chemical shifts of the ferriheme methyl groups were used to predict orientations of the axial ligand. The obtained axial ligand plane angle φ is consistent with that from the molecular dynamics simulation but not with those from the crystal data. Compared with mouse neuroglobin, the obtained average ligand orientation of human neuroglobin reflects the changeability of heme environment for the Ngb family.     

The reactivity of cytochrome c with soft ligands

The spectral changes caused by binding soft ligands to the cytochrome c iron and their correlation to ligand affinities support the hypothesis that the iron—methionine sulfur bond of this heme protein is enhanced by delocalization of the metal l₂, electrons into the empty 3d orbitals of the ligand atom. These findings also explain the unique spectrum of cytochrome c in the far red.     

Effects of extrinsic imidazole ligation on the molecular and electronic structure of cytochrome c

Although imidazole ligand binding to cytochrome c is not directly related to its physiological function, it has the potential to provide valuable information on the molecular and electronic structure of the protein. The solution structure of the imidazole adduct of oxidized horse heart cytochrome c (Im-cyt c) has been determined through 2D NMR spectroscopy. The Im-cyt c, 8 mM in 1.2 M imidazole solution at pH 5.7 and 313 K, provided altogether 2,542 NOEs (1,901 meaningful NOEs) and 194 pseudocontact shifts. The 35 conformers of the family show the RMSD values to the average structure of 0.063+/-0.007 nm for the backbone and 0.107+/-0.007 nm for all heavy atoms, respectively. The characterization of Im-cyt c is discussed in detail both in terms of structure and electronic properties. The replacement of the axial ligand Met80 with the exogenous imidazole ligand induces significant conformation changes in both backbone and side chains of the residues located in the distal axial ligand regions. The imidazole ligand binds essentially parallel to the imidazole of the proximal histidine, the two planes forming an angle of 8+/-7 degrees. The electron delocalization on the heme moiety and the magnetic susceptibility tensor are consistent with these structural features.     

1H nmr studies of complexes of ferric leghemoglobin with substituted pyridines and nicotinic acids

The ¹H nuclear magnetic resonance (nmr) spectra of complexes of soybean ferric leghemoglobin with 3-substituted pyridines and 5-substituted nicotinic acids have been recorded in order to determine the influence of axial ligands on heme electronic structure. The hyperfine shifted resonances of the heme group were assigned by analogy to previous assignments for the pyridine and nicotinic acid complexes of leghemoglobin. The spectra are characteristic of predominantly low-spin ferric heme complexes. For the pyridine complexes, the rate of ligand exchange was found to increase with decreasing ligand pK_A. For many of the complexes, optical and nmr spectra reveal the presence of an equilibrium mixture of high- and low-spin states of the iron atom. The percentage of high-spin component increases with decreasing ligand pK_A Smaller hyperfine shifts are noted for leghemoglobin complexes with ligands capable of weak ligand → metal π bonding. The pattern of hyperfine shifted resonances is similar for all complexes studied and indicates that the overall heme electronic structure is dominated by the bonding to the proximal histidine.     

The use of pseudocontact shifts to refine solution structures of paramagnetic metalloproteins: Met80Ala cyano-cytochrome c as an example

 The availability of NOE constraints and of the relative solution structure of a paramagnetic protein permits the use of pseudocontact shifts as further structural constraints. We have developed a strategy based on: (1) determination of the χ tensor anisotropy parameters from the starting structure; (2) recalculation of a new structure by using NOE and pseudocontact shift constraints simultaneously; (3) redetermination of the χ tensor anisotropy parameters from the new structure, and so on until self-consistency. The system investigated is the cyanide derivative of a variant of the oxidized Saccharomyces cerevisiae iso-1-cytochrome c containing the Met80Ala mutation. The structure has been substantially refined. It is shown that the analysis of the deviation of the experimental pseudocontact shifts from those calculated using the starting structure may be unsound, as may the simple structure refinement based on the pseudocontact shift constraints only. Received: 11 July 1995 / Accepted: 30 October 1995     

An NMR structural study of nickel-substituted rubredoxin

The Ni(II) and Zn(II) derivatives of Desulfovibrio vulgaris rubredoxin (DvRd) have been studied by NMR spectroscopy to probe the structure at the metal centre. The βCH₂ proton pairs from the cysteines that bind the Ni(II) atom have been identified using 1D nuclear Overhauser enhancement (NOE) difference spectra and sequence specifically assigned via NOE correlations to neighbouring protons and by comparison with the published X-ray crystal structure of a Ni(II) derivative of Clostridium pasteurianum rubredoxin. The solution structures of DvRd(Zn) and DvRd(Ni) have been determined and the paramagnetic form refined using pseudocontact shifts. The determination of the magnetic susceptibility anisotropy tensor allowed the contact and pseudocontact contributions to the observed chemical shifts to be obtained. Analysis of the pseudocontact and contact chemical shifts of the cysteine Hβ protons and backbone protons close to the metal centre allowed conclusions to be drawn as to the geometry and hydrogen-bonding pattern at the metal binding site. The importance of NH–S hydrogen bonds at the metal centre for the delocalization of electron spin density is confirmed for rubredoxins and can be extrapolated to metal centres in Cu proteins: amicyanin, plastocyanin, stellacyanin, azurin and pseudoazurin.     

Rubredoxin as a paramagnetic relaxation-inducing probe

The paramagnetic effect due to the presence of a metal center with unpaired electrons is no longer considered a hindrance in protein NMR spectroscopy. In the present work, the paramagnetic effect due to the presence of a metal center with unpaired electrons was used to map the interface of an electron transfer complex. Desulfovibrio gigas cytochrome c₃ was chosen as target to study the effect of the paramagnetic probe, Fe-rubredoxin, which produced specific line broadening in the heme IV methyl resonances M2¹ and M18¹. The rubredoxin binding surface in the complex with cytochrome c₃ was identified in a heteronuclear 2D NMR titration. The identified heme methyls on cytochrome c₃ are involved in the binding interface of the complex, a result that is in agreement with the predicted complexes obtained by restrained molecular docking, which shows a cluster of possible solutions near heme IV. The use of a paramagnetic probe in ¹HNMR titration and the mapping of the complex interface, in combination with a molecular simulation algorithm proved to be a valuable strategy to study electron transfer complexes involving non-heme iron proteins and cytochromes.     

Structure of cytochrome c551 from Pseudomonas aeruginosa refined at 1.6 Å resolution and comparison of the two redox forms

The molecular structures of ferri- and ferrocytochrome c₅₅₁ from Pseudomonas aeruginosa have been refined at a resolution of 1.6 Å, to an R factor of 19.5% for the oxidized molecule and 18.7% for the reduced. Reduction of oxidized crystals with ascorbate produced little change in cell dimensions, a 10% mean change in F_obs, and no damage to the crystals. The heme iron is not significantly displaced from the porphyrin plane. Bond lengths from axial ligands to the heme iron are as expected in a low-spin iron compound. A total of 67 solvent molecules were incorporated in the oxidized structure, and 73 in the reduced, of which four are found inside the protein molecule. The oxidized and reduced forms have virtually identical tertiary structures with 2 ° root-mean-square differences in main-chain torsion angles φ and ψ, but with larger differences along the two edges of the heme crevice. The difference map and pyrrole ring tilt suggest that a partially buried water molecule (no. 23) in the heme crevice moves upon change of oxidation state.Pseudomonas cytochrome c₅₅₁ differs from tuna cytochrome c in having: (1) a water molecule (no. 23) at the upper left of the heme crevice; that is, between Pro62 and the heme pyrrol 3 ring on the sixth ligand Met61 side, where tuna cytochrome c has an evolutionary invariant Phe82 ring; (2) a string of hydrophobic side-chains along the left side of the heme crevice, and fewer positively charged lysines in the vicinity; and (3) a more exposed and presumably more easily ionizable heme propionate group at the bottom of the molecule. A network of hydrogen bonds in the heme crevice is reminiscent of that inside the heme crevice of tuna cytochrome c. As in tuna, a slight motion of the water molecule toward the heme is observed in the oxidized state, helping to give the heme a more polar microenvironment. The continuity of solvent environment between the heme crevice and the outer medium could explain the greater dependence of redox potential on pH in cytochrome c₅₅₁ than in cytochrome c.     

Stanniocalcin 1 binds hemin through a partially conserved heme regulatory motif

Hemin (iron protoporphyrin IX) is a necessary component of many proteins, functioning either as a cofactor or an intracellular messenger. Hemoproteins have diverse functions, such as transportation of gases, gas detection, chemical catalysis and electron transfer. Stanniocalcin 1 (STC1) is a protein involved in respiratory responses of the cell but whose mechanism of action is still undetermined. We examined the ability of STC1 to bind hemin in both its reduced and oxidized states and located Cys¹¹⁴ as the axial ligand of the central iron atom of hemin. The amino acid sequence differs from the established (Cys–Pro) heme regulatory motif (HRM) and therefore presents a novel heme binding motif (Cys–Ser). A STC1 peptide containing the heme binding sequence was able to inhibit both spontaneous and H₂O₂ induced decay of hemin. Binding of hemin does not affect the mitochondrial localization of STC1.