Controlling the regiospecificity and coupling of cytochrome P450cam: T185F mutant increases coupling and abolishes 3-hydroxynorcamphor product.

Cytochrome P450cam (P450CIA1) catalyzes the hydroxylation of camphor and several substrate analogues such as norcamphor and 1-methyl-norcamphor. Hydroxylation was found experimentally at the 3, 5, and 6 positions of norcamphor, but only at the 5 and 6 positions of 1-methyl-norcamphor. In the catalytic cycle, the hydroxylation of substrate is coupled to the consumption of NADH. For camphor, the degree of coupling is 100%, but for both norcamphor and 1-methyl-norcamphor, the efficiency is dramatically lowered to 12% and 50%, respectively. Based on an examination of the active site of P450cam, it appeared that mutating position 185 might dramatically alter the product specificity and coupling of hydroxylation of norcamphor by P450cam. Analysis of molecular dynamics trajectories of norcamphor bound to the T185F mutant of cytochrome P450cam predicted that hydroxylation at the 3 position should be abolished and that the coupling should be dramatically increased. This mutant was constructed and the product profile and coupling experimentally determined. The coupling was doubled, and hydroxylation at the 3 position was essentially abolished. Both of these results are in agreement with the prediction.

Analysis of active site motions from a 175 picosecond molecular dynamics simulation of camphor-bound cytochrome P450cam.

The structure and internal motions of the active site residues of camphor-bound cytochrome P450cam have been evaluated on the basis of a 175 psec molecular dynamics simulation. The active site residues generally show very small deviations away from their starting crystal positions. These residues also generally show much smaller fluctuations than for the enzyme as a whole. Phe 87 is dynamically very unusual and is suggested to play a role in substrate movement into and/or out of the active site. The average distance between the heme iron and atoms C5, C6, and C3 of camphor is 5.3, 6.0, and 7.0 A, respectively. This trend is consistent with the experimentally observed stereospecificity of the hydroxylation reaction. On the basis of distance and angle criteria, both 5-exo and 5-endo hydrogen abstraction are predicted to occur during the hydroxylation reaction; although the 5-exo pathway is expected to be 3-fold more likely.

3 Nsec molecular dynamics simulation of the protein ubiquitin and comparison with X-ray crystal and solution NMR structures.

Mainly due to computational limitations, past protein molecular dynamics simulations have rarely been extended to 300 psec; we are not aware of any published results beyond 350 psec. The present work compares a 3000 psec simulation of the protein ubiquitin with the available x-ray crystallographic and solution NMR structures. Aside from experimental structure availability, ubiquitin was studied because of its relatively small size (76 amino acids) and lack of disulfide bridges. An implicit solvent model was used except for explicit treatment of waters of crystallization. We found that the simulated average structure retains most of the character of the starting x-ray crystal structure. In two highly surface accessible regions, the simulation was not in agreement with the x-ray structure. In addition, there are six backbone-backbone hydrogen bonds that are in conflict between the solution NMR and x-ray crystallographic structures; two are bonds that the NMR does not locate, and four are ones that the two methods disagree upon the donor. Concerning these six backbone-backbone hydrogen bonds, the present simulation agrees with the solution NMR structure in five out-of-the six cases, in that if a hydrogen bond is present in the x-ray structure and not in the NMR structure, the bond breaks within 700 psec. Of the two hydrogen bonds that are found in the NMR structure and not in the x-ray structure, one forms at 1400 psec and the other forms rarely. The present results suggest that relatively long molecular dynamics simulations, that use protein x-ray crystal coordinates for the starting structure and a computationally efficient solvent representation, may be used to gain an understanding of conformational and dynamic differences between the solid-crystal and dilute-solution states.

Substrate mobility in thiocamphor-bound cytochrome P450cam: an explanation of the conflict between the observed product profile and the X-ray structure.

Thiocamphor is an unusual substrate for P450cam in that in the X-ray structure it binds in the active site pocket in two distinct orientations and neither of these orientations are consistent with the 5-alcohol being the primary product. Other camphor analogs such as norcamphor or camphane bind in a single orientation consistent with the 5-alcohol being a major product. We present an analysis of four 175 ps molecular dynamics trajectories of thiocamphor-bound cytochrome P450cam. The first two trajectories were calculated for cytochrome P450cam with thiocamphor bound in both its major and minor crystallographic orientations. In the second set of simulations, a single oxygen atom was added as a distal ligand to the heme group in order to model the putative ferryl oxygen reaction intermediate. Trajectories were again calculated starting with thiocamphor in its major and minor orientations. While the protein dynamics were quite similar in all four trajectories, the substrate showed distinctly different motions in each of the trajectories. In particular, the preferred substrate orientations were very different in the presence of the ferryl oxygen than in the absence of that oxygen. The preferred orientations in the absence of the distal oxygen were consistent with the 3-alcohol being the major product, while the preferred orientations in the presence of the distal oxygen were consistent with the 5-alcohol being a major product. These simulations offer an explanation for the inconsistency between the X-ray data and the product profile.

Dramatic differences in the motions of the mouth of open and closed cytochrome P450BM-3 by molecular dynamics simulations.

Molecular dynamics trajectories were calculated separately for each of the two molecules in the asymmetric unit of the crystal structure of the hemoprotein domain of cytochrome P450BM-3. Each simulation was 200 ps in length and included a 10 A layer of explicit solvent. The simulated time-average structure of each P450BM-3 molecule is closer to its crystal structure than the two molecular dynamics time-averaged structures are to each other. In the crystal structure, molecule 2 has a more accessible substrate binding pocket than molecule 1, and this difference is maintained throughout the simulations presented here. In particular, the substrate docking regions of molecule 1 and molecule 2 diverge in the solution state simulations. The mouth of the substrate binding pocket is significantly more mobile in the simulation of molecule 2 than in the simulation of molecule 1. For molecule 1, the width of the mouth is only slightly larger than its X-ray value of 8.7 A and undergoes fluctuations of about 1 A. However, in molecule 2, the mouth of the substrate binding pocket is dramatically more open in the time-average molecular dynamics structure (14.7 A) than in the X-ray structure (10.9 A). Furthermore, this region of the protein undergoes large amplitude motions during the trajectory that are not seen in the trajectory of molecule 1, repeatedly opening and closing up to 7 A. Presumably, the binding of different substrates will induce the mouth region to adopt different conformations from within the wide range of structures that are accessible.

Active-site mobility inhibits reductive dehalogenation of 1,1,1-trichloroethane by cytochrome P450cam.

Recent studies by Wackett and co-workers have shown that cytochrome P450cam is capable of reductively dehalogenating hexachloroethane at a significant rate, but that no appreciable dehalogenation of 1,1,1-trichloroethane is observed. A growing body of evidence indicates that differences in intrinsic reactivity can not completely explain this observation. We therefore explored the possible role of differences in preferred binding orientation and in active-site mobility. A detailed analysis of molecular dynamics trajectories with each of these substrates bound at the active site of P450cam is presented. While the dynamics and overall time-average structure calculated for the protein are similar in the two trajectories, the two substrates behave quite differently. The smaller substrate, 1,1,1-trichloroethane, is significantly more mobile than hexachloroethane and has a preferred orientation in which the substituted carbon is generally far from the heme iron. In contrast, for hexachloroethane, one of the chlorine atoms is nearly always in van der Waals contact with the heme iron, which should favor the initial electron transfer step.

Binding free energy calculations for P450cam-substrate complexes.

A recently proposed semi-empirical method for calculating binding free energies was used to examine the binding of a variety of substrates to cytochrome P450cam. For a set of 11 different potential substrates of cytochrome P450cam, both the absolute and relative binding free energies were generally well reproduced. The mean error in the calculated absolute binding free energy for all 11 compounds is 0.55 kcal/mol. Forty-eight out of 55 calculated relative binding free energies have the correct sign and the mean unsigned error between calculated and experimental relative binding free energies is 0.77 kcal/mol. For one substrate, thiocamphor, the effect of substrate orientation on the calculated binding free energy was examined. The ability of this method to predict the effect of active site mutations was also examined in two cases.

A 175-psec molecular dynamics simulation of camphor-bound cytochrome P-450cam.

The structure and internal motions of cytochrome P-450cam, a monooxygenase heme enzyme with 414 amino acid residues, with camphor bound at the active site have been evaluated on the basis of a 175-psec molecular dynamics simulation carried out at 300 K. All hydrogen atoms were explicitly modeled, and 204 crystallographic waters were included in the simulation. Based on an analysis of the time course of the trajectory versus potential energy, root mean square deviation, radius of gyration, and hydrogen bonding, the simulation was judged to be stable and representative of the average experimental structure. The averaged structural properties of the enzyme were evaluated from the final 135 psec of the simulation. The average atomic displacement from the X-ray structure was 1.39 A for all heavy atoms and 1.17 A for just C-alpha atoms. The average root-mean-square (rms) fluctuations of all heavy atoms and backbone atoms were 0.42 and 0.37 A, respectively. The computed rms fluctuations were in reasonable agreement with the experimentally determined temperature factors. All 13 segments of alpha-helix and 5 segments of beta-sheet were well preserved with the exception of the N-terminal half of helix F which alternated between an alpha-helix and a 310-helix. In addition there were in general only small variations in the relative orientation of adjacent alpha-helices. The rms fluctuations of the backbone dihedral angles in the secondary structure elements were almost uniformly smaller, with the fluctuation in alpha-helices and beta-sheets, 31 and 10% less, respectively, than those in nonsecondary structure regions. The reported crystal structure contains kinks in both helices C and I. In the simulation, both of these regions showed high mobility and large deviations from their starting positions. Since the kink in the I helix is at the oxygen binding site, these motions may have mechanistic implications.

Predicting the product specificity and coupling of cytochrome P450cam.

We present an analysis of several molecular dynamics trajectories of substrate-bound cytochrome P450cam. Trajectories were calculated for the native substrate, camphor, as well as for the alternative substrates, norcamphor and thiocamphor. The system modeled consisted of the crystallographically resolved amino acids, the heme group with a single oxygen atom as the distal ligand, the bound substrate, and the crystallographic waters. These trajectories of the presumptive ferryl oxygen intermediate were used to predict regiospecificity of hydroxylation and coupling between NADH consumption and product formation. Simple geometric criteria in combination with electronic considerations were used to calculate the probability of hydroxylation at specific sites on the substrate. We found that for all the cases examined, the predicted product ratios were in good agreement with the experimentally observed values. We also determined that these simple geometric criteria can be used to predict the degree of coupling between NADH consumption and product formation for a given substrate, which was in good agreement with the experimental values.

Molecular modeling of mammalian cytochromes P450: application to study enzyme function.

Cytochromes P450 are important heme-containing enzymes that catalyze the oxidation of a vast array of endogenous and exogenous compounds, including drugs and carcinogens. One of the more successful approaches to study P450 function involves molecular modeling. Because none of the mammalian P450s have been crystallized, a number of homology models have been constructed based on the structures of known bacterial P450s. Molecular models, generated using molecular replacement or distance geometry methods, can be used to dock substrates and/or inhibitors in the active site to explain various aspects of enzyme function. The majority of modeling research has dealt with enzyme-substrate interactions in the active site. The analysis of these interactions has helped us to better understand the mechanism of P450 catalysis and provided the structural basis for the regio- and stereospecificity of substrate oxidation as well as susceptibility to inhibition or inactivation. The models have been utilized to identify and/or confirm key residues and to rationally interpret experimental data. The alteration in activity in a mutant P450 can be related to changes in enzyme-substrate/inhibitor interactions, such as the removal or appearance of van der Waals overlaps or changes in compound mobility. Homology models can also help to analyze P450-redox partner interactions and identify critical determinants of protein stability. We can expect further development of molecular modeling methods and their increasing contribution into research on P450 function as an integral part of a combined theoretical-experimental approach.

Substrate mobility in a deeply buried active site: analysis of norcamphor bound to cytochrome P-450cam as determined by a 201-psec molecular dynamics simulation.

While cytochrome P-450cam catalyzes the hydroxylation of camphor to 5-exo-hydroxycamphor with 100% stereospecificity, norcamphor is hydroxylated by this enzyme yielding 45% 5-exo-, 47% 6-exo-, and 8% 3-exo-hydroxynorcamphor (Atkins, W.M., Sligar, S.G., J. Am. Chem. Soc. 109:3754-3760, 1987). The present study describes a 201-psec molecular dynamics (MD) stimulation of norcamphorbound cytochrome P-450cam to elucidate the relationship between substrate conformational mobility and formation of alternative products. First, these data suggest that the product specificity is, at least partially, due to the mobility of the substrate within the active site. Second, the high mobility of norcamphor in the active site leads to an average increase in separation between the heme iron and the substrate of about 1.0 A; this increase in separation may be the cause of the uncoupling of electron transfer when norcamphor is the substrate. Third, the active site water located in the norcamphorbound crystal structure possesses mobility that correlates well with the spin-state equilibrium of this enzyme-substrate complex.

Ethylbenzene hydroxylation by cytochrome P450cam.

The metabolism of ethylbenzene by cytochrome P450cam was analyzed by experimental and theoretical methods. The present experiments indicate that ethylbenzene is hydroxylated almost exclusively at the secondary ethyl carbon with about a 2:1 ratio of R:S product. Several molecular dynamics trajectories were performed with different starting conformations of ethylbenzene in the active site of P450cam. The stereochemistry of hydroxylation predicted from the molecular dynamics simulations was found to be in good agreement with the observed products.

Stereoselective hydroxylation of norcamphor by cytochrome P450cam. Experimental verification of molecular dynamics simulations.

The stereoselectivity of cytochrome P450cam hydroxylation has been investigated with the enantiomerically pure substrate analog norcamphor. (1R)- and (1S)-norcamphor (> 92 enantiomeric excess) were characterized in the hydroxylation reaction with cytochrome P450cam with respect to the product profile, steady state kinetics, coupling efficiency, and free energy of substrate dissociation. The experimental results demonstrate regiospecificity that is enantiomer-specific and confirm our previously reported prediction that (1R)-norcamphor is hydroxylated preferentially at the 5-carbon and (1S)-norcamphor at the 6-carbon (Bass, M. B., and Ornstein, R. L. (1993) J. Comput. Chem. 14, 541-548); these simulation results are now compared with simulations involving a ferryl oxygen intermediate. Hydroxylation of (1R)-norcamphor was found at the 5-, 6-, and 3-carbons in a ratio of 65:30:5 (respectively), whereas (1S)-norcamphor was oxidized to produce a 28:62:10 ratio of the same products. With the exception of the regiospecificity, all of the reaction and physical parameters are similar for each enantiomer of norcamphor. These results show that the position of the carbonyl group on the hydrocarbon skeleton of norcamphor plays a role in determining the average orientation of this substrate in the active site and suggests that hydrogen bonding interactions can aid in directing the regiospecificity and stereospecificity of the hydroxylation reaction catalyzed by cytochrome P450cam.