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1 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 Extracting quantitative structural information from EPR g-tensors with density functional theory: applications to nitrosoiron(II) porphyrins S. Patchkovskii and T. Ziegler Department of Chemistry, University of Calgary, 2500 University Dr. NW, Calgary, Alberta, T2N 1N4 Canada I am on the Web: http://www.cobalt.chem.ucalgary.ca/ps/posters/EPR-FeP/

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2 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 Introduction Conclusions and outlookConclusions and outlook Introduction.Introduction. Iron-containing porphyrin complexes (hemes) serve as prosthetic groups in many vitally important enzymes [1,2], such as hemoglobin. Because oxy-hemoglobin possesses a singlet electronic structure, it is not amenable to studies using electron paramagnetic resonance (EPR). Structurally similar heme nitrosyls have a spatially non- degenerate spin-doublet ground state, which is readily observable with EPR. Experimental EPR spectra of nitrosylated hemoglobins and myoglobins [3,4], indicate the presence of two distinct radical species: rhombic and axial. The rhombic ("Type I") species exhibits three distinct principal components in its EPR g tensor (g 1 =1.96-1.98, g 2 =2.00, g 3 =2.06-2.08). It has been assigned to a six-coordinated structure, with NO coordinated in a bent end-on orientation, and the second axial position filled by an imidazole side chain. Despite extensive investigations, the nature of the axial ("Type II") species (g || =1.99-2.00, g =2.02- 2.03) have proven more elusive, and remains controversial. In this work, we examine the structure and EPR g-tensors of model porphyrins with density functional theory (DFT). On the basis of our calculations, we propose a new structural model for the axial species.

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3 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 Theory Quasi-relativistic DFT formulation of the EPR g-tensors used in this work distinguishes between several contributions to the g-tensor [5,6] : free-electron g value ( 2.0023) diamagnetic termparamagnetic term kinetic energy correction mass-velocity correction Darwin term Relativistic corrections The paramagnetic term dominates deviation of g from the free-electron value for complexes considered here, and can be in turn separated into several contributions: frozen core contributions occupied-occupied coupling terms occupied-virtual coupling terms The occ-vir term is usually the most qualitatively important contribution. Theory ITheory I

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4 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 The contribution is given by (atomic units): 0.00731 the effective potential -spin current due to unit magnetic field along s=x,y, or z -spin current The form of the occupied-virtual paramagnetic contribution the the EPR g-tensor is analogous to the expression for the paramagnetic part of the NMR shielding tensor for a nucleus N, given by: The similarity between the two quantities is extremely useful both in the evaluation and in analysis of g tensor, and is unique to our DFT implementation. Theory IITheory II

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5 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 The spin-current density for a spin arising due to the coupling between occupied and virtual MOs caused by the external magnetic field B 0 is given by: magnetic coupling coefficient unperturbed occupied MOunperturbed virtual MO field strength in the direction s (=x,y,z) The principal contribution to the coupling coefficient u is in turn given by: unperturbed orbital energiesunperturbed MO coefficients atomic orbitals (AOs) applies to each AO Theory IIITheory III

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6 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 Methods MethodsMethods

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7 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 ON-Fe(P): Coordination of NO Conclusions and outlookConclusions and outlook ON-Fe(P): Coordination of NOON-Fe(P): Coordination of NO 1a: +3.9 kcal/mol 1b: +0.0 kcal/mol 1c: +0.0 kcal/mol The coordination of NO around iron gives rise to three key points on the PES of 1, namely: the C 4v structure 1a with the linearly coordinated NO ligand.This is a second-order saddle point, rather than a minimum. C s structure 1b, with bent end-on coordination of the NO ligand, pointing towards one of the meso carbon atoms of the porphyrin ligand. a second C s structure 1c, with the NO bond eclipsing one of the equatorial Fe-N p bonds. Given the isoenergetic 1a and 1b, the NO ligand in free five-coordinated heme nitrosyl should undergo free intramolecular rotation around the Z axis. A closer inspection of the optimised structures reveals that the rotation of the NO ligand is coupled to the distortion of the porphyrin ligand. As a consequence, hindering the distortion increases the barrier for NO rotation increases to about 1.2 kcal/mol. Z

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8 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 ON-Fe(P)-Im: Coordination of imidazole Conclusions and outlookConclusions and outlook ON-Fe(P)-Im: Coordination of imidazoleON-Fe(P)-Im: Coordination of imidazole 1.0 2.0 3.0 2.02.22.42.62.8 E, kcal/mol R Fe-N(Im),Å Dissociation of the imidazole was examined by gradually increasing the iron-imidazole distance, and optimizing the rest of the structure. The resulting energy profile is exceptionally flat, with variations in the Fe-N(Im) bond length in the 2.05-2.50Å range changing energy by less than 1 kcal/mol. As a consequence, experimental bond lengths are likely to be influenced by substitution and environment effects. Although the potential energy profile for the dissociation of imidazole appears to indicate a presence of a local minimum at 2.4Å, g-tensors show no qualitative changes in the vicinity of this structure. exp

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9 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 Conclusions and outlookConclusions and outlook ON-Fe(P)-Im: rotation of the axial ligandsON-Fe(P)-Im: rotation of the axial ligands 32 2a2b2c2d2d 2e2e 0.00 0.20 0.40 0.60 050100150200250300350 NO, degree Im = 0º Im =45º energy, kcal/mol In the six-coordinated complex, both axial ligands preferentially appear in staggered orientation (2a-2c). The local minima appear within 0.3 kcal/mol of the global minimum (2c), and will be substantially populated at non-zero temperature. The orientations of NO and imidazole in condensed phases are likely to be determined by substituent effects and intermolecular interactions. Free rotation of both axial substituents may also be expected at room temperature.

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10 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 Origin of the g tensors: C 4v structure Conclusions and outlookConclusions and outlook Origin of g in C4v structure.Origin of g in C4v structure. +3.0 +2.0 +1.0 0.0 -2.0 -3.0 -4.0 eV +14 +20 -36 1*1* 22 11 n1n1 SOMO -spin -spin Contributions to g are in parts per thousand (ppt) Mulliken d-population on iron in 1 ( 6.7) is consistent with d 7 electron count, formally making 1 a complex of Fe I and NO +. The unpaired electron is mostly on iron’s d z 2 AO. The SOMO vanishes upon action of the M z operator, so that all spin-restricted terms in the parallel component g || also vanish. If the magnetic field is in the XY plane, the field- induced coupling with -spin * MOs (d xz, d yz -like), localised on the Fe-N=O fragment, results in a contribution of -36 ppt to g . This contribution is almost identically cancelled by two -spin terms, (+14 and +20 ppt)

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11 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 Conclusions and outlookConclusions and outlook Origin of g in meso-Cs structureOrigin of g in meso-Cs structure Origin of the g tensors: C s structures +3.0 +2.0 +1.0 0.0 -2.0 -3.0 -4.0 eV +14 +20 -36 1*1* 22 11 n1n1 +37 +7 +32 -15 -13 linear bent In the bent 1b, iron's d z 2 AOs overlap with the * orbitals of the NO ligand, tilting the d z 2 - like contribution to the SOMO towards the direction perpendicular to the N=O bond. Such orientation allows SOMO to interact with occupied non-bonding d x 2 -y 2 orbital n 1. The SOMO and SOMO n terms exhibit a qualitatively different dependence on the relative orientation of the NO ligand and the porphyrin core, so that g-tensor in bent 1 depends on the orientation of NO.

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12 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 Origin of the g tensors: ON-Fe(P)-Im Conclusions and outlookConclusions and outlook Origin of g in ON-Fe(P)-ImOrigin of g in ON-Fe(P)-Im 1b: SOMO 2b: SOMO Upon coordination of the imidazole, a weak * interaction between an imidazole lone pair, and the d z 2 -like lobe of the SOMO, destabilises the SOMO by 0.9 eV. The Mulliken spin population on iron is also reduced, from 0.9 (1b) to 0.5 (2b). decrease in the d character of the SOMO reduces all spin- orbit coupling matrix elements, decreasing all g ’s. a large NO * contribution to the SOMO increases the magnitude of matrix elements of the M operator in -SOMO - * terms. destabilisation of the SOMO increases -SOMO - * terms, while decreasing -SOMO - contributions.

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13 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 g1g1 g2g2 g3g3 1a2.0082.003 1b1.9942.0052.063 1c1.9972.0242.033 ON-Fe(TPP)2.0102.0642.102 2a1.9551.9952.034 2d1.9672.0002.012 ON-Fe(TPP)-Im1.9702.0032.072 Mb(NO)1.9792.0022.076 -Hb(NO) 1.9742.0062.081 -Hb(NO) 1.9782.0082.057 Numerical results: Principal components Conclusions and outlookConclusions and outlook Numerical results: Principal componentsNumerical results: Principal components *: TPP = tetraphenylporphyrinato 2- ; Mb = myoglobin; Hb = hemoglobin As expected from the qualitative analysis, calculated principal g-tensor components in ON-Fe(P) (1) are sensitive to both the Fe-N=O bond angle, and to the orientation of the NO relative to the porphyrin (1a- 1c). The values are only in a broad qualitative agreement with experiment, and appear to indicate an orientation of the NO ligand intermediate between 1b and 1c. Part of the error may be due to the neglect of the environment effects. In ON-Fe(P)-Im (2), g-tensor is again sensitive to the orientation of NO, but is left unchanged by imidazole rotation. Calculated magnitudes of the principal components appear to be in a broad qualitative agreement with experiment. The characteristic g 1

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14 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 Conclusions and outlookConclusions and outlook Numerical results: g-tensor orientationsNumerical results: g-tensor orientations Numerical results: g tensor orientation g3g3 g1g1 g2g2 g2g2 g1g1 g free Theoretical orientations of the g-tensor components in 2 are in a good agreement with experiment for low-temperature "Type I" nitroso myoglobin. At 77K, the principal components of the g tensor in MbNO [3] deviate from the direction perpendicular to porphyrin plane (Z axis) by, respectively, 63±2º (g 1 ), 27±2º (g 2 ), and 88±2º (g 3 ). The corresponding theoretical values for free 2a are 58º, 32º, and 90º. Comparison of the remaining experimental direction cosines with the theoretical results gives of approximately -70º. The good agreement in the theoretical and experimental orientations of the g- tensor indicated that the {FeNO} fragment in MbNO is not substantially distorted by the protein. The large g 2 orientation has been taken previously [4] as an indication of the Fe-N(NO) axial bond direction. As can be seen from the SOMO plots, it in fact corresponds to the direction of the d z 2 -like part of the SOMO, which in turn deviates from the Fe-N(NO) bond by almost 40º.

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15 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 Conclusions and outlookConclusions and outlook Numerical results: RFe-N(Im) and g-tensorNumerical results: RFe-N(Im) and g-tensor Numerical results: R Fe-N(Im) and g-tensor Elongation of the Fe-N(Im) bond results in an increase in all three principal components. The free electron-like component (g 2 ) increases by only 8 ppt/Å in the 2.0- 2.9 Å range. The other two components, g 1 and g 3, increase at the rates of, respectively, 45 and 36 ppt/Å. At the separation of 2.9 Å or more, the calculated g tensor becomes essentially identical to the result for free five-coordinated species. If the NO ligand rotates freely on the EPR time scale, averaging results in g > g ||. The parallel component increases the rate of 19 ppt/Å. g grows at 35 ppt/Å, so that the separation between the rotationally averaged components increases with dissociation of imidazole. g tensor components

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16 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 Biological ON-Fe(P)-Im: Models galore Conclusions and outlookConclusions and outlookConclusions and outlookConclusions and outlook Biological ON-Fe(P)-Im: Models galoreBiological ON-Fe(P)-Im: Models galoreBiological ON-Fe(P)-Im: Models galoreBiological ON-Fe(P)-Im: Models galore bound-dissociated AxialRhombicSource linear- bent Model up- down inclined- straight [3] [13] [15] [16] P.E.S.g-tensor Axial structure is not a minimum; axial g-tensor components are wrong Rhombic structure is not a minimum; “Axial” structure has rhombic g-tensor. “Axial” structure has rhombic g-tensor. “Axial” structure is not a minimum, and has rhombic g-tensor

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17 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 Biological ON-Fe(P)-Im: Rhombic species Conclusions and outlookConclusions and outlook Biological ON-Fe(P)-Im: rhombic speciesBiological ON-Fe(P)-Im: rhombic species The rhombic signal ("State I") exhibits a characteristic pattern of g min

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18 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 Biological ON-Fe(P)-Im: Axial species Conclusions and outlookConclusions and outlook Biological ON-Fe(P)-Im: axial speciesBiological ON-Fe(P)-Im: axial species The axial (“Type II”) spectra are usually found at higher temperatures. In the absence of a satisfactory static model, it appears reasonable to consider possible dynamical interpretations. If the rotation of NO is unfreezed, averaging of the g components leads to an axial spectrum. In the model complex, imidazole has no additional chemical bonds to the ON-Fe(P) fragment, and will dissociate completely. In heme proteins, it is provided by a histidine residue, and is tethered to the backbone. The tethering may prevent a complete dissociation, leading to smaller values of g and g ||. It also allows protein to switch between Type I and Type II spectra at the same temperature: A conformational change in the backbone can pull the imidazole from the rest of the complex, g || gg Calculated2.002.03 -Hb(NO) 2.002.02 -Hb(NO) 2.002.03 effectively causing its dissociation. This would, in turn, lower the barrier for NO rotation, and produce the change in the spectrum.

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19 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 Summary Conclusions and outlookConclusions and outlook SummarySummary Structure of ON-Fe(P)-Im: In both five- and six-coordinated complexes, NO is preferably coordinated end-on, with a Fe-N=O bond angle of approximately 140º. In the gas-phase five-coordinated complex, NO undergoes free rotation. Imidazole coordination in the second axial position increases the activation barrier Variations in Fe-N(Im) bond in the 2.05-2.5Å change energy by less than 1 kcal/mol. EPR g-tensors: are sensitive to the orientation of NO and Fe-N(Im) bond length. are not sensitive to the orientation of the imidazole ligand orientation of the g-tensor component, showing the smallest deviation from the free- electron value, does not coincide with the direction of the axial Fe-N(NO) bond. Biological ON-Fe(P)-Im systems: Rhombic ("Type I") EPR signal in nitrosoheme systems corresponds to a static structure with NO in a staggered orientation, and R Fe-N(Im) 2.1Å. The ON-Fe(P) moiety is not noticeably distorted by the protein environment. The axial ("Type II") EPR signal is tentatively assigned to a partially dissociated species (R Fe-N(Im) 2.5Å), with a freely rotating NO ligand.

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20 Structure of heme nitrosyls from EPR g-tensors with DFT CSC’83, 2000 Acknowledgements AcknowledgementsAcknowledgements This work has been supported by the National Sciences and Engineering Research Council of Canada (NSERC), as well as by the donors of the Petroleum Research Fund, administered by the American Chemical Society (ACS-PRF No 31205-AC3). Dr. Georg Schreckenbach is gratefully acknowledged for making the GIAO- DFT implementation of the EPR g tensors available to the authors. References

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