Evidence for Stabilization of the Low-Spin State of Cytochrome P450

Sheffield S10 2JF, U.K., and ML Laboratories plc, 60 London Road, St. Albans AL1 1NG, U.K.. Received January 26, 1998. The cytochrome P450 superfamily...
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Chem. Res. Toxicol. 1998, 11, 962-966

Evidence for Stabilization of the Low-Spin State of Cytochrome P450 Due to Shortening of the Proximal Heme Bond Matthew D. Segall,*,† Mike C. Payne,† Wynne Ellis,‡ Geoff T. Tucker,‡ and Nick Boyes§ Cavendish Laboratory (TCM), University of Cambridge, Cambridge CB3 0HE, U.K., Department of Medicine and Pharmacology, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, U.K., and ML Laboratories plc, 60 London Road, St. Albans AL1 1NG, U.K. Received January 26, 1998

The cytochrome P450 superfamily of enzymes is ubiquitous, being responsible for the metabolism of a wide range of endogenous and xenobiotic compounds. However, the detailed mechanism of the catalytic cycle of these enzymes is still not fully understood. We describe results, obtained from first principles molecular simulations, which indicate that the low-spin state of the Fe3+ ion, present in the heme moiety at the active site of a cytochrome P450 enzyme, may be stabilized by shortening of the proximal bond of the heme. Calculations indicate that a bond length of less than ∼2.05 Å between the heme Fe3+ ion and the cysteine S, which forms the proximal ligand, would result in the stabilization of the low-spin state of the Fe3+, inhibiting the progress of the P450 catalytic cycle. Our investigation uses novel first principles modeling techniques which treat the entire system quantum-mechanically.

Introduction The P450 superfamily of enzymes catalyze the oxygenation of a wide variety of hydrophobic substrates. Understanding the action of these enzymes is important due to their participation in the metabolism of endogenous compounds and in the activation/deactivation of a wide range of xenobiotics including drugs (1). P450s are hemoproteins made up of between 400 and ∼500 amino acids and containing a single heme prosthetic group. The proximal axial ligand of this heme is formed by a cysteine residue. The catalytic reaction occurs at the heme iron ion and generally results in the hydroxylation of a hydrophobic substrate molecule. The catalytic cycle is summarized in Figure 1. Our investigation focuses on the system between processes 1 and 2, after the substrate has bound but before the first reduction. Fe3+ (ferric iron) can exist in three spin states, S ) 1/2, S ) 3/5, or S ) 5/2. In biological systems Fe3+ is usually found in either the S ) 1/2, low-spin (LS)1 state, in which the five 3d electrons are maximally paired, or S ) 5/2, high-spin (HS) state, in which the five 3d electrons are maximally unpaired. In the heme system, 6-fold coordinated Fe3+ is generally found to be LS and 5-fold coordinated Fe3+ is found in the HS state. The nature of the axial heme ligands have an important effect on the LS-HS balance. A strong axial field will bring about a relatively large d-orbital splitting, favoring the LS state. For example, the axial ligand field strength due to imidazolate is much stronger than that due to cysteinate †

University of Cambridge. University of Sheffield. ML Laboratories plc. 1 Abbreviations: P450 cam, cytochrome P450 CYP101 derived from Pseudomonas putida; LS, low spin; HS, high spin. ‡ §

Figure 1. Cytochrome P450 reaction cycle. R and ROH represent substrate and hydroxylated substrate, respectively. The states shown in a dotted box have not been directly observed.

(2). It can be calculated that a change of axial ligand from thiolate to imidazole in a ferroheme-CO complex will result in a shift of the UV absorption band due to a d f d transition from about 450 to 420 nm (3). Ligand-free P450 exhibits a Soret absorption maximum in a UV spectrum at approximately 420 nm. This is associated with the LS state of the Fe3+. Electronic spectral (4), NMR (5), and crystallographic (6) data indicate that a water molecule forms a sixth axial ligand of the Fe3+ in the substrate-free form, thus stabilizing the LS state of the ion (See Figure 2). The binding of a substrate is identified by a reduction in the Soret absorption band at 420 nm and a corresponding increase in a maximum at 390 nm. This is indicative of a change from a LS to HS state of the ferric iron. X-ray crystallography of substrate-bound complexes of the P450cam (P450 CYP101) enzyme (7) shows that the

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Stabilization of the P450 Low-Spin State

Figure 2. Heme complex of ligand-free P450cam. Note the water molecule that forms the sixth axial ligand of the heme iron. Carbon atoms are shown as bonds only; other atom types are labeled. Hydrogen atoms have been omitted except in the case of the iron-ligated water molecule.

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did not produce a change from the LS to a HS state. This suggested that a mechanism other than the displacement of the sixth ligand was responsible for the stabilization of the LS state. The mechanism identified was a reduction in the length of the bond between the Fe3+ and cysteine S from 2.4 Å in the unbound enzyme to 2.0 Å in the camphane-bound system. In this article we confirm that the shortening of this bond is responsible for the favorability of the LS state of the by studying another substrate analogue-bound system (norcamphor). We also demonstrate that artificial shortening of the Fe3+-S bond when a substrate (camphor) is bound to P450cam leads to the LS state being favored. Furthermore, the calculation of the ground-state spin as a function of bond length provides an estimate for the minimum Fe3+-S bond length which favors the HS state and hence allows the catalytic reaction to proceed efficiently.

Experimental Section

Figure 3. Fragment of the norcamphor-bound system modeled. The norcamphor substrate (top) and fragments of the heme moiety and iron-ligated cysteine residue (bottom) are shown. Note the water molecule that remains coordinated with the iron atom. The full heme moiety and cysteine residue were included in the computational models. Carbon atoms are shown as bonds only; other atom types are labeled. Hydrogen atoms have been omitted except in the case of the iron-ligated water molecule.

iron-ligated water molecule is displaced in these cases, changing the Fe3+ from a 6-fold to a 5-fold coordination state. The Fe3+ ion is also seen to move out of the plane of the heme. The change from a LS to HS state is usually correlated with a lowering of the redox potential. For example, in P450cam the redox potential changes from -300 mV in the absence of a ligand to -173 mV in the presence of camphor. This makes the first reduction of the Fe3+ energetically favorable, and the reaction cycle proceeds. Substrate analogues of P450cam differ from true substrates in that they are either smaller or do not form a hydrogen bond with the Tyr 96 residue in the active site. This allows substrate analogues greater freedom of movement within the active site. The crystal structures of complexes of substrate analogues with P450cam show that the Fe3+-coordinated water molecule is retained (810) and a high fraction of the LS character of the remains. An example of the heme complex at the active site of a substrate analogue-bound P450 may be seen in Figure 3. The presence of the water is thought to be the primary cause of the stabilization of the LS state (11). The redox potentials of the substrate analogue-bound complexes fall between those of the substrate-free and substrate-bound systems. Indeed, a linear free energy relationship between the redox potential and spin state has been found (12). In ref 13 we presented preliminary results from computational simulations, demonstrating that removal of the Fe3+-coordinated water molecule from the active site of P450cam bound to camphane (a substrate analogue)

Ab initio, or first principles, molecular modeling methods solve the quantum mechanical equations which govern the behavior of a system. The only information which has to be provided are the atomic numbers and positions of the atoms within the system. In contrast, empirical or semiempirical approaches require a model of the interactions between the atoms to be supplied. The parameters of these models are usually derived by fitting the outcome of simulations to experimental data. The problems with these techniques arise when you consider the question of the range of their applicability. If the parameters of the models were derived from system A, what guarantee is there that they apply to system B? A number of approximations must be made in order to perform ab initio calculations on all but the smallest of systems. However, these approximations are based on general physical principles and do not rely on the specific nature of the system under investigation. Thus, we can have great confidence in the results of “computational experiments” as there is never a problem about the assumptions of the model being violated by the changes made during an investigation. These “computational experiments” can have many advantages over their conventional brethren. They offer a very high degree of control over experimental conditions; a single parameter may be changed and the result observed. In addition, an enormous range of chemical and physical observables may be calculated with only one piece of “apparatus”, the computer. The careful use of computational modeling can substantially aid in our understanding of biological processes. The ab initio methods we employ are described in detail in refs 13 and 15. The dominant cost of conventional quantum chemistry calculations lies in the accurate treatment of the electron-electron interactions. We address this problem by using the Kohn-Sham (16) formulation of density functional theory (17) with a spin-dependent generalized gradient approximation for the exchange-correlation potential (18). The electronic wave functions were represented using a plane wave basis set with a cut off energy of 600 eV. The use of a plane wave basis set would be prohibitively expensive if the core electrons were included in the calculation. Therefore, only the valence electrons were treated explicitly with the core electrons and nuclei represented by optimized pseudopotentials (19). Nonlinear core corrections were applied for the Fe core, as we found these to be necessary to model the spin behavior of transition metals correctly (20). The use of a plane wave basis set requires the system to be periodic. Therefore, the supercell approximation was used whereby the system is repeated periodically in space separated by a large vacuum region. Interactions between charged periodic images were accounted for using a post-hoc correction (21). The charge and spin on the Fe atom was calculated by Mulliken population analysis (22)

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Segall et al.

after projection of the plane wave eigenstates on to a localized basis set of atomic pseudo-orbitals (23). As the full structure of a P450 enzyme is prohibitively large to be modeled with an ab initio approach, we include only the heme moiety, cysteine residue, and ligand molecule in our simulations. A previous study of the determinants of the spin state of P450cam by Harris and Loew (24), using the semiempirical INDO method, indicated that the electrostatic field of the protein must be included in calculations performed on such a limited fragment of the enzyme in order to explain the resting spin state of the Fe3+. In contrast, results obtained using our ab initio approach (14) indicate that this fragment is sufficient to accurately reproduce the spin properties of the Fe3+ without explicit consideration of the direct effect of the remaining protein. Of course, the structure of the surrounding protein is of great importance in binding the ligand molecule, which determines the orientation of the molecule relative to the heme Fe3+, and in modifying the geometry of the heme-cysteine complex. The use of high-resolution crystal structures of P450cam in complex with a number of different ligands (8, 9, 25) allows the geometries of the heme complex in each system to be accurately reproduced in our models. The Brookhaven Protein Database references of the P450 crystal structures used in the simulations described in this article were 7CPP (norcamphor complex), 4CP4 (camphor complex), and 6CPP (camphane complex).

Results and Discussion The results in ref 13 indicated that the removal of the Fe3+-coordinated water molecule from the camphanebound P450cam active site, leaving the remaining geometry unchanged, did not lead to a change from a LS to HS state of the Fe3+. To test the generality of this finding for substrate analogue-bound systems, the same experiment was performed on the norcamphor-bound system shown in Figure 3. The results of these calculations, shown in Figure 4A, also demonstrated no change in the ground state spin of the Fe3+. This indicates that there is another mechanism maintaining the LS character of the system. Examination of the geometry of the system indicates that the Fe3+-S bond length is significantly shorter than that found in the ligand-free and camphorbound crystal structures (2.4 Å). If the length of the bond is increased in our models and the calculations repeated, as shown in Figure 4B, we find that the removal of the water does lead to a clear change from LS to HS. This is consistent with the accepted view that the Fe3+coordinated water must be removed for the system to adopt a HS character, but it also confirms the previous finding that the shortening of the Fe3+-S bond in these substrate analogue-bound complexes is responsible for the stabilization of the LS state. The most probable cause of the decrease in the length of the Fe-S bond may be seen if we examine the charge on the Fe3+ ion in the unmodified ligand-bound complexes. In the ligand-bound systems, in which the Fe3+ ion has moved out of the plane of the heme, we can see that the presence of an Fe3+-coordinated water molecule causes an increase in the charge by approximately 0.1e. As the cysteine S atom is negatively charged, this will cause an increase in the attraction between these ions. Therefore, we see that the presence of the water in these substrate analogue-bound systems probably leads to the energetic favorability of the LS state of the Fe3+ but that the LS state of the Fe3+ is not a direct effect of the presence of the water as postulated in the standard model of the interaction.

Figure 4. Schematic diagrams of the norcamphor systems modeled, with the results of the spin and charge on the Fe3+. Results are given for the systems with and without an Fe3+coordinated water molecule. (A) shows the system in its unmodified state; in (B) the Fe-S bond has been stretched to 2.4 Å. The overlap populations between the Fe and S atoms are shown in italics. Spins are given in units of p, and charges, in e. The plane of the heme moiety is represented by a dashed line.

It is desirable to determine if this phenomenon is restricted to substrate analogue-bound systems. Therefore, calculations for the spin on the Fe3+ were performed for the natural camphor-bound complex and for this system with the Fe3+-S bond artificially reduced to 2 Å (the bond length found in the crystal structure of the camphane-bound complex). The results of these calculations, shown in Figure 5, correctly assign a high-spin value in the natural camphor-bound system, as camphor is a substrate of P450cam. Furthermore, the ground-state spin is found to take a LS value on reducing the Fe3+-S bond length. This indicates that the effect of shortening the proximal bond is not specific to the substrate analoguebound complexes. To understand the chemical origin of the behavior noted above, the strength of bonding between the Fe and S can be compared in the different systems by examining the overlap populations between these atoms. These values are also shown in Figures 4 and 5 and indicate that the Mulliken overlap populations of the shorter Fe3+-S bonds are approximately 10% higher than the longer bonds. This indicates that the shortening of the bond causes an increase in the bonding interaction between the Fe and S atoms. As described above, it is understood that a strong axial field causes an increase in splitting of the d-orbitals, favoring the LS state. The Fe3+-S bond length at which the spin changes from LS to HS in the absence of an Fe3+-coordinated water molecule may be predicted by performing calculations of the spin for a range of different bond lengths. The spin is plotted in Figure 6 against bond length for the camphane-bound system in the absence of an Fe3+coordinated water molecule. This shows a sharp change between the LS and HS state at a bond length of approximately 2.05 Å. This is in good agreement with

Stabilization of the P450 Low-Spin State

Figure 5. Schematic diagrams of the camphor systems modeled, with the results of the spin and charge on the Fe3+. (A) shows the system in its unmodified state; in (B) the Fe3+-S bond has been reduced to 2.0 Å. The overlap populations between the Fe and S atoms are shown in italics. Spins are given in units of p, and charges, in e. The plane of the heme moiety is represented by a dashed line.

Figure 6. Graph of the spin on the Fe3+ ion against the Fe3+-S bond length in the camphane-bound system, with no Fe3+coordinated water molecule. Calculated points are shown as 4’s. Two values are plotted for a bond length of 2.05 Å as the highand low-spin states differ in energy by only 0.2 kcal/mol, compared with the value of kT at physiological temperatures of 0.6 kcal/mol. Therefore, the thermodynamic average of the spins of these states is plotted as O. The difference in energy between the high- and low-spin states for other bond lengths is much larger so that no significant thermodynamic mixing will occur.

experimental evidence. Comparison of crystal structures of substrate-bound complexes (camphor (25) and adamantanone (9)) with those of substrate analogue-bound structures (camphane (8), norcamphor (9), and thiocamphor (8)) shows that the average Fe3+-S bond length in the substrate-bound cases is 2.26 Å whereas in the substrate analogue complexes it is 2.04 Å. The fraction of high-spin state found experimentally for the substrates lies between 96% and 98%, while those for the substrate analogues vary between 46% and 65% (8). In particular,

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the adamantanone complex of P450cam is found to adopt a >95% HS state (12). The crystal structure of this complex (9) shows the Fe3+-S bond length is 2.11 Å, significantly shorter than that found in the camphor complex but above the crossover point found in these calculations. It should be noted that the results given apply only to the ground state of the system and do not include any thermodynamic effects which result in a significant mixture of high spin character, even in the systems in which the ground state is LS. One result of this is the experimental observation that norcamphor is significantly hydroxylated, with an efficiency of 12%, even though the system has only a 46% HS character (8), and the results we obtain indicate a LS ground state (13). We calculate the internal energy difference between the LS and HS states of the Fe3+ in the model system to be 2.96 kcal mol-1. If a thermal distribution is used to calculate the fraction of time spent in the HS state, neglecting dynamic and entropic effects, we obtain a result of 11%. If we assume that the reduction occurs when the Fe3+ is in a HS state, this is consistent with the experimental observations. The norcamphor complex exhibits an Fe3+-S bond length of 2.05 Å, very close to the crossover point indicated for the camphane-bound system. Our calculations indicate that this system has a strictly LS character, even in the absence of an Fe3+-ligated water molecule. This is consistent with our findings as there are small differences between the norcamphor- and camphanebound systems which may serve to further reinforce the LS character of the in the former. Very close to the crossover point in the energy surface between the LS and HS states, at an Fe3+-S bond length of approximately 2.05 Å, the spin will be very sensitive to differences in geometry of the heme that affect the environment of the ion. It is well understood that the proportion of HS and LS states can be modified in 5-fold coordinated ferric porphyrin compounds with an axial σ-bonded carbon by varying the nature of the σ-bonded ligand (see for example refs 26-28). We have shown that such an effect may be predicted in P450 using ab initio modeling techniques. In particular, our simulations provide strong evidence for the stabilization of the low-spin state of the heme by a proximal Fe3+-S bond length shorter than approximately 2.05 Å. P450 enzymes with a proximal bond length close to 2.05 Å may have particularly interesting properties. In the absence of an Fe3+coordinated water, the spin on the Fe3+ will be very sensitive to changes in the geometry of the active site. Thus, the efficiency of hydroxylation of different ligands with such an enzyme may vary significantly, even if they all displace the Fe3+-coordinated water on binding.

Acknowledgment. M.D.S. acknowledges the financial support of ML Laboratories plc. The calculations presented in this article were performed on the Hitachi SR2201 located at the University of Cambridge High Performance Computing Facility and the Hitachi SR2001 located at Hitachi Europe’s Maidenhead (United Kingdom) headquarters.

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