3site Multisubstrate-Bound State of Cytochrome P450cam.

We identified a multisubstrate-bound state, hereby referred as a 3site state, in cytochrome P450cam via integrating molecular dynamics simulation with nuclear magnetic resonance (NMR) pseudocontact shift measurements. The 3site state is a result of simultaneous binding of three camphor molecules in three locations around P450cam: (a) in a well-established "catalytic" site near heme, (b) in a kink-separated "waiting" site along channel-1, and (c) in a previously reported "allosteric" site at E, F, G, and H helical junctions. These three spatially distinct binding modes in the 3site state mutually communicate with each other via homotropic allostery and act cooperatively to render P450cam functional. The 3site state shows a significantly superior fit with NMR pseudo contact shift (PCS) data with a Q-score of 0.045 than previously known bound states and consists of D251 free of salt-bridges with K178 and R186, rendering the enzyme functionally primed. To date, none of the reported cocomplex of P450cam with its redox partner putidaredoxin (pdx) has been able to match solution NMR data and controversial pdx-induced opening of P450cam's channel-1 remains a matter of recurrent discourse. In this regard, inclusion of pdx to the 3site state is able to perfectly fit the NMR PCS measurement with a Q-score of 0.08 and disfavors the pdx-induced opening of channel-1, reconciling previously unexplained remarkably fast hydroxylation kinetics with a koff of 10.2 s-1. Together, our findings hint that previous experimental observations may have inadvertently captured the 3site state as an in vitro solution state, instead of the catalytic state alone, and provided a distinct departure from the conventional understanding of cytochrome P450.

Redox partner recognition and selectivity of cytochrome P450lin (CYP111A1).

The strict requirement of cytochrome P450cam for its native ferredoxin redox partner, putidaredoxin (Pdx), is not exhibited by any other known cytochrome P450 (CYP) system and the molecular details of redox partner selectivity are still not completely understood. We therefore examined the selectivity of a related Pseudomonas cytochrome P450, P450lin, by testing its activity with non-native redox partners. We found that P450lin could utilize Arx, the native redox partner of CYP101D1, to enable turnover of its substrate, linalool, while Pdx showed limited activity. Arx exhibited a higher sequence similarity to P450lins native redox partner, linredoxin (Ldx) than Pdx, including several residues that are believed to be at the interface of the two proteins, based on the P450cam-Pdx complex structure. We therefore mutated Pdx to resemble Ldx and Arx and found that a double mutant, D38L/∆106, displayed higher activity than Arx. In addition, Pdx D38L/∆106 does not induce a low-spin shift in linalool bound P450lin but does destabilize the P450lin-oxycomplex. Together our results suggest that P450lin and its redox partners may form a similar interface to P450cam-Pdx, but the interactions that allow for productive turnover are different.

Solution phase refinement of active site structure using 2D NMR and judiciously 13C-labeled cytochrome P450.

The Cytochrome P450 (CYP450) superfamily has been the subject of intense research for over six decades. Here the HU227 strain of E. coli, lacking the δ-aminolevulinic acid (δ-ALA) synthase gene, was employed, along with [5-13C] δ-ALA, in the heterologous expression of P450cam harboring a prosthetic group labeled with 13C at the four methine carbons (Cm) and pyrrole Cα positions. The product was utilized as a proof of principle strategy for defining and refining solution phase active site structure in cytochrome P450cam, providing proton-to-proton distances from 13CmH to protons on bound substrate or nearby amino acid residues, using short mixing time 2D or 3D NOESY-HMQC methods. The results reveal the interesting finding that 2D 13C-filtered NOESY-HMQC can be used to obtain distances between protons on labeled 13C to positions of protons nearby in the active site, confirming the utility of this NMR-based approach to probing active site structure under physiological conditions. Such 13C-heme-filtered NOE data complement X-ray crystallographic and T1-based NMR measurements; and, may also be of potentially significant utility in furnishing experimental distance constraints in validations of docking routines commonly employed for determining the relative affinities and binding orientations of drug candidates with CYP450s.

Hydroxylation Regiochemistry Is Robust to Active Site Mutations in Cytochrome P450cam (CYP101A1).

Cytochrome P450cam (CYP101A1) catalyzes the hydroxylation of d-camphor by molecular oxygen. The enzyme-catalyzed hydroxylation exhibits a high degree of regioselectivity and stereoselectivity, with a single major product, d-5-exo-hydroxycamphor, suggesting that the substrate is oriented to facilitate this specificity. In previous work, we used an elastic network model and perturbation response scanning to show that normal deformation modes of the enzyme structure are highly responsive not only to the presence of a substrate but also to the substrate orientation. This work examines the effects of mutations near the active site on substrate localization and orientation. The investigated mutations were designed to promote a change in substrate orientation and/or location that might give rise to different hydroxylation products, while maintaining the same carbon and oxygen atom balances as in the wild type (WT) enzyme. Computational experiments and parallel in vitro site-directed mutations of CYP101A1 were used to examine reaction products and enzyme activity. 1H-15N TROSY-HSQC correlation maps were used to compare the computational results with detectable perturbations in the enzyme structure and dynamics. We found that all of the mutant enzymes retained the same regio- and stereospecificity of hydroxylation as the WT enzyme, with varying degrees of efficiency, which suggests that large portions of the enzyme have been subjected to evolutionary pressure to arrive at the appropriate sequence-structure combination for efficient 5-exo hydroxylation of camphor.

Caught in the act: Monitoring OO bond cleavage in Acylperoxoferric cytochrome P450cam to form compound I in real time.

While monitoring the reaction of ferric cytochrome P450cam (Cyp101) with substituted peroxybenzoic acids using rapid-scanning, stopped-flow (RSSF) spectroscopy, an intermediate appears en route to formation of the high-valent moiety known as Compound I [Fe(IV)=O/porphyrin radical cation] that is thought to be the key catalytic species for O-atom transfer to substrate. We have previously suggested (Spolitak, T., Dawson, J.H., Ballou, D.P., J. Biol. Chem.2005, 280, 20,300-20,309) that this species is an acylperoxo-ferric heme adduct that subsequently undergoes OO bond cleavage to generate Compound I. Singular value decomposition analysis of the RSSF data for formation of this intermediate shows that the energy of its Soret absorption peak is sensitive to the electron donor properties of the aryl substituents on the peracid. A linear Hammett correlation plot is seen for the energy of the Soret absorption peak vs. the Hammett σ constant. This correlation requires that the aryl substituents remain as part of the ligand bound to the heme iron, providing direct evidence that the adduct is indeed a ferric acylperoxo derivative. Linear Hammett correlation plots are also seen for both the rate of formation of the intermediate as well as for its conversion to Compound I. It is proposed that the electron donating/withdrawing properties of the aryl-bound substituents affect the electrophilic nature for binding substrate, changing the observed rate of formation for the acylperoxo intermediate, as well as the propensity and stability of the substituted benzoic acid to serve as the leaving group during OO bond cleavage yielding Compound I.

Determination of Multidirectional Pathways for Ligand Release from the Receptor: A New Approach Based on Differential Evolution.

Steered molecular dynamics (SMD) simulation is a powerful method in computer-aided drug design as it can be used to access the relative binding affinity with high precision but with low computational cost. The success of SMD depends on the choice of the direction along which the ligand is pulled from the receptor-binding site. In most simulations, the unidirectional pathway was used, but in some cases, this choice resulted in the ligand colliding with the complex surface of the exit tunnel. To overcome this difficulty, several variants of SMD with multidirectional pulling have been proposed, but they are not completely devoid of disadvantages. Here, we have proposed to determine the direction of pulling with a simple scoring function that minimizes the receptor-ligand interaction, and an optimization algorithm called differential evolution is used for energy minimization. The effectiveness of our protocol was demonstrated by finding expulsion pathways of Huperzine A and camphor from the binding site of Torpedo California acetylcholinesterase and P450cam proteins, respectively, and comparing them with the previous results obtained using memetic sampling and random acceleration molecular dynamics. In addition, by applying this protocol to a set of ligands bound with LSD1 (lysine specific demethylase 1), we obtained a much higher correlation between the work of pulling force and experimental data on the inhibition constant IC50 compared to that obtained using the unidirectional approach based on minimal steric hindrance.

Updating the Paradigm: Redox Partner Binding and Conformational Dynamics in Cytochromes P450.

This Account summarizes recent findings centered on the role that redox partner binding, allostery, and conformational dynamics plays in cytochrome P450 proton coupled electron transfer. P450s are one of Nature's largest enzyme families and it is not uncommon to find a P450 wherever substrate oxidation is required in the formation of essential molecules critical to the life of the organism or in xenobiotic detoxification. P450s can operate on a remarkably large range of substrates from the very small to the very large, yet the overall P450 three-dimensional structure is conserved. Given this conservation of structure, it is generally assumed that the basic catalytic mechanism is conserved. In nearly all P450s, the O2 O-O bond must be cleaved heterolytically enabling one oxygen atom, the distal oxygen, to depart as water and leave behind a heme iron-linked O atom as the powerful oxidant that is used to activate the nearby substrate. For this process to proceed efficiently, externally supplied electrons and protons are required. Two protons must be added to the departing O atom while an electron is transferred from a redox partner that typically contains either a Fe2S2 or FMN redox center. The paradigm P450 used to unravel the details of these mechanisms has been the bacterial CYP101A1 or P450cam. P450cam is specific for its own Fe2S2 redox partner, putidaredoxin or Pdx, and it has long been postulated that Pdx plays an effector/allosteric role by possibly switching P450cam to an active conformation. Crystal structures, spectroscopic data, and direct binding experiments of the P450cam-Pdx complex provide some answers. Pdx shifts the conformation of P450cam to a more open state, a transition that is postulated to trigger the proton relay network required for O2 activation. An essential part of this proton relay network is a highly conserved Asp (sometimes Glu) that is known to be critical for activity in a number of P450s. How this Asp and proton delivery networks are connected to redox partner binding is quite simple. In the closed state, this Asp is tied down by salt bridges, but these salt bridges are ruptured when Pdx binds, leaving the Asp free to serve its role in proton transfer. An alternative hypothesis suggests that a specific proton relay network is not really necessary. In this scenario, the Asp plays a structural role in the open/close transition and merely opening the active site access channel is sufficient to enable solvent protons in for O2 protonation. Experiments designed to test these various hypotheses have revealed some surprises in both P450cam and other bacterial P450s. Molecular dynamics and crystallography show that P450cam can undergo rather significant conformational gymnastics that result in a large restructuring of the active site requiring multiple cis/trans proline isomerizations. It also has been found that X-ray driven substrate hydroxylation is a useful tool for better understanding the role that the essential Asp and surrounding residues play in catalysis. Here we summarize these recent results which provide a much more dynamic picture of P450 catalysis.

Nanopore-based measurement of the interaction of P450cam monooxygenase and putidaredoxin at the single-molecule level.

Protein-protein interactions occur in a wide range of biological processes and are of great significance to life function. Characterization of transient protein-protein interactions remains a significant barrier to our understanding of cellular processes. Nanopores provide unique nanoscale environments that accommodate single molecules from the surrounding bulk solution. This method permits label-free sensing at the single-molecule level with extremely high sensitivity. Herein, the interaction between a single P450cam monooxygenase and its redox partner putidaredoxin (Pdx) was monitored via transient ionic current by using functionalized glass nanopores. Results show that the volume of P450cam determines the blockage current while the interactions between the P450cam and Pdx give a long blockage duration. Our glass nanopore sensor with adjustable diameter could be applied for real-time sensing of protein-protein interactions between individual proteins with a wide range of molecular weight.

A Conserved Allosteric Site on Drug-Metabolizing CYPs: A Systematic Computational Assessment.

Cytochrome P450 enzymes (CYPs) are the largest group of enzymes involved in human drug metabolism. Ligand tunnels connect their active site buried at the core of the membrane-anchored protein to the surrounding solvent environment. Recently, evidence of a superficial allosteric site, here denoted as hotspot 1 (H1), involved in the regulation of ligand access in a soluble prokaryotic CYP emerged. Here, we applied multi-scale computational modeling techniques to study the conservation and functionality of this allosteric site in the nine most relevant mammalian CYPs responsible for approximately 70% of drug metabolism. In total, we systematically analyzed over 44 µs of trajectories from conventional MD, cosolvent MD, and metadynamics simulations. Our bioinformatic analysis and simulations with organic probe molecules revealed the site to be well conserved in the CYP2 family with the exception of CYP2E1. In the presence of a ligand bound to the H1 site, we could observe an enlargement of a ligand tunnel in several members of the CYP2 family. Further, we could detect the facilitation of ligand translocation by H1 interactions with statistical significance in CYP2C8 and CYP2D6, even though all other enzymes except for CYP2C19, CYP2E1, and CYP3A4 presented a similar trend. As the detailed comprehension of ligand access and egress phenomena remains one of the most relevant challenges in the field, this work contributes to its elucidation and ultimately helps in estimating the selectivity of metabolic transformations using computational techniques.

Controlling the Substrate Specificity of an Enzyme through Structural Flexibility by Varying the Salt-Bridge Density.

Many enzymes, particularly in one single family, with highly conserved structures and folds exhibit rather distinct substrate specificities. The underlying mechanism remains elusive, the resolution of which is of great importance for biochemistry, biophysics, and bioengineering. Here, we performed a neutron scattering experiment and molecular dynamics (MD) simulations on two structurally similar CYP450 proteins; CYP101 primarily catalyzes one type of ligands, then CYP2C9 can catalyze a large range of substrates. We demonstrated that it is the high density of salt bridges in CYP101 that reduces its structural flexibility, which controls the ligand access channel and the fluctuation of the catalytic pocket, thus restricting its selection on substrates. Moreover, we performed MD simulations on 146 different kinds of CYP450 proteins, spanning distinct biological categories including Fungi, Archaea, Bacteria, Protista, Animalia, and Plantae, and found the above mechanism generally valid. We demonstrated that, by fine changes of chemistry (salt-bridge density), the CYP450 superfamily can vary the structural flexibility of its member proteins among different biological categories, and thus differentiate their substrate specificities to meet the specific biological needs. As this mechanism is well-controllable and easy to be implemented, we expect it to be generally applicable in future enzymatic engineering to develop proteins of desired substrate specificities.

Partial Opening of Cytochrome P450cam (CYP101A1) Is Driven by Allostery and Putidaredoxin Binding.

Cytochrome P450cam (CYP101A1) catalyzes the regio- and stereo-specific 5-exo-hydroxylation of camphor via a multistep catalytic cycle that involves two-electron transfer steps, with an absolute requirement that the second electron be donated by the ferrodoxin, putidaredoxin (Pdx). Whether P450cam, once camphor has bound to the active site and the substrate entry channel has closed, opens up upon Pdx binding, during the second electron transfer step, or it remains closed is still a matter of debate. A potential allosteric site for camphor binding has been identified and postulated to play a role in the binding of Pdx. Here, we have revisited paramagnetic NMR spectroscopy data and determined a heterogeneous ensemble of structures that explains the data, provides a complete representation of the P450cam/Pdx complex in solution, and reconciles alternative hypotheses. The allosteric camphor binding site is always present, and the conformational changes induced by camphor binding to this site facilitates Pdx binding. We also determined that the state to which Pdx binds comprises an ensemble of structures that have features of both the open and closed state. These results demonstrate that there is a finely balanced interaction between allosteric camphor binding and the binding of Pdx at high camphor concentrations.

Active Site Hydrogen Bonding Induced in Cytochrome P450cam by Effector Putidaredoxin.

Cytochrome P450s are diverse and powerful catalysts that can activate molecular oxygen to oxidize a wide variety of substrates. Catalysis relies on effective uptake of two electrons and two protons. For cytochrome P450cam, an archetypal member of the superfamily, the second electron must be supplied by the redox partner putidaredoxin (Pdx). Pdx also plays an effector role beyond electron transfer, but after decades the mechanism remains under investigation. We applied infrared spectroscopy to heme-ligated CN- to examine the influence of Pdx binding. The results indicate that Pdx induces the population of a conformation wherein the CN- ligand forms a strong hydrogen bond to a solvent water molecule, experimentally corroborating the formation of a proposed proton delivery network. Further, characterization of T252A P450cam implicates the side chain of Thr252 in regulating the population equilibrium of hydrogen-bonded states within the P450cam/Pdx complex, which could underlie its role in directing activated oxygen toward product formation and preventing reaction uncoupling through peroxide release.

Covalent conjugation of single-walled carbon nanotube with CYP101 mutant for direct electrocatalysis.

Covalent linkage between the single-walled carbon nanotube (SWCNT) and CYP101 through a specific site of the enzyme can provide a novel method of designing efficient enzyme electrodes using this prototype cytochrome P450 enzyme. We have chemically modified the SWCNT with linker 4-carboxy phenyl maleimide (CPMI) containing maleimide functional groups. The enzyme was covalently attached on to the SWCNT through the maleimide group of the linker (CPMI) to the thiolate group of the surface exposed Cys 58 or Cys 136 of the CYP101 forming a covalently immobilized protein on the nanotube. Thin film of the modified SWCNT-CPMI-CYP101conjugate was made on a glassy carbon (GC) electrode. Direct electrochemistry of the substrate (camphor)-bound enzyme was studied using this immobilized enzyme electrode system and the redox potential was found to be -320mV vs Ag/AgCl (3 M KCl), which agrees with the redox potential of the substrate bound enzyme reported earlier. The electrochemically driven enzymatic mono-oxygenation of camphor by this immobilized enzyme electrode system was studied by measurement of the catalytic current at different concentrations of camphor. The catalytic current was found to increase with increasing concentration of camphor in presence of oxygen. The product formed during the catalysis was identified by mass-spectrometry as hydroxy-camphor.

Reconciling conformational heterogeneity and substrate recognition in cytochrome P450.

Cytochrome P450, the ubiquitous metalloenzyme involved in detoxification of foreign components, has remained one of the most popular systems for substrate-recognition process. However, despite being known for its high substrate specificity, the mechanistic basis of substrate-binding by archetypal system cytochrome P450cam has remained at odds with the contrasting reports of multiple diverse crystallographic structures of its substrate-free form. Here, we address this issue by elucidating the probability of mutual dynamical transition to the other crystallographic pose of cytochrome P450cam and vice versa via unbiased all-atom computer simulation. A robust Markov state model, constructed using adaptively sampled 84-µs-long molecular dynamics simulation trajectories, maps the broad and heterogenous P450cam conformational landscape into five key substates. In particular, the Markov state model identifies an intermediate-assisted dynamic equilibrium between a pair of conformations of P450cam, in which the substrate-recognition sites remain "closed" and "open," respectively. However, the estimate of a significantly higher stationary population of closed conformation, coupled with faster rate of open → closed transition than its reverse process, dictates that the net conformational equilibrium would be swayed in favor of "closed" conformation. Together, the investigation quantitatively infers that although a potential substrate of cytochrome P450cam would, in principle, explore a diverse array of conformations of substrate-free protein, it would mostly encounter a "closed" or solvent-occluded conformation and hence would follow an induced-fit-based recognition process. Overall, the work reconciles multiple precedent crystallographic, spectroscopic investigations and establishes how a statistical elucidation of conformational heterogeneity in protein would provide crucial insights in the mechanism of potential substrate-recognition process.

Dynamics underlying hydroxylation selectivity of cytochrome P450cam.

Structural heterogeneity and the dynamics of the complexes of enzymes with substrates can determine the selectivity of catalysis; however, fully characterizing how remains challenging as heterogeneity and dynamics can vary at the spatial level of an amino acid residue and involve rapid timescales. We demonstrate the nascent approach of site-specific two-dimensional infrared (IR) spectroscopy to investigate the archetypical cytochrome P450, P450cam, to better delineate the mechanism of the lower regioselectivity of hydroxylation of the substrate norcamphor in comparison to the native substrate camphor. Specific locations are targeted throughout the enzyme by selectively introducing cyano groups that have frequencies in a spectrally isolated region of the protein IR spectrum as local vibrational probes. Linear and two-dimensional IR spectroscopy were applied to measure the heterogeneity and dynamics at each probe and investigate how they differentiate camphor and norcamphor recognition. The IR data indicate that the norcamphor complex does not fully induce a large-scale conformational change to a closed state of the enzyme adopted in the camphor complex. Additionally, a probe directed at the bound substrate experiences rapidly interconverting states in the norcamphor complex that explain the hydroxylation product distribution. Altogether, the study reveals large- and small-scale structural heterogeneity and dynamics that could contribute to selectivity of a cytochrome P450 and illustrates the approach of site-selective IR spectroscopy to elucidate protein dynamics.

Proton Relay Network in the Bacterial P450s: CYP101A1 and CYP101D1.

Cytochrome P450s are among nature's most powerful catalysts. Their ability to activate molecular dioxygen to form high-valent ferryl intermediates (Compounds I and II) enables a wide array of chemistries ranging from simple epoxidations to more complicated C-H bond oxidations. Oxygen activation is achieved by reduction of the ferrous dioxygen complex, which requires the transfer of an electron from a redox partner and subsequent double protonation to yield a water molecule and a ferryl porphyrin π-cation radical (Compound I). Previous studies of the CYP101 family of cytochrome P450s demonstrated the importance of the conserved active site Asp25X residue in this protonation event, although its precise role is yet to be unraveled. To further explore the origin of protons in oxygen activation, we analyzed the effects of an Asp to Glu mutation at the 25X position in P450cam and in CYP101D1. This mutation inactivates P450cam but not CYP101D1. A series of mutagenic, crystallographic, kinetic, and molecular dynamics studies indicate that this mutation locks P450cam into a closed, inactive conformation. In CYP101D1, the D259E mutant changes the rate-limiting step to reduction of the P450-oxy complex, thus opening a window into the critical proton-coupled electron transfer step in P450 catalysis.

Unexpected Differences between Two Closely Related Bacterial P450 Camphor Monooxygenases.

The bacterial cytochrome P450cam catalyzes the oxidation of camphor to 5-exo-hydroxycamphor as the first step in the oxidative assimilation of camphor as a carbon/energy source. CYP101D1 is another bacterial P450 that catalyzes the same reaction. A third P450 (P450tcu) has recently been discovered that has ≈86% sequence identity to P450cam as well as very similar enzymatic properties. P450tcu, however, exhibits three unusual features not found in P450cam. First, we observe product in at least two orientations in the X-ray structure that indicates that, unlike the case for P450cam, X-ray-generated reducing equivalents can drive substrate hydroxylation in crystallo. We postulate, on the basis of molecular dynamics simulations, that greater flexibility in P450tcu enables easier access of protons to the active site and, together with X-ray driven reduction, results in O2 activation and substrate hydroxylation. Second, the characteristic low-spin to high-spin transition when camphor binds occurs immediately with P450cam but is very slow in P450tcu. Third, isothermal titration calorimetry shows that in P450cam substrate binding is entropically driven with a ΔH of >0 while in P450tcu with a ΔH of <0 with a more modest change in -TΔS. These results indicate that despite nearly identical structures and enzymatic properties, these two P450s exhibit quite different properties most likely related to differences in conformational dynamics.

Proton relay network in P450cam formed upon docking of putidaredoxin.

Cytochromes P450 are versatile heme-based enzymes responsible for vital life processes. Of these, P450cam (substrate camphor) has been most studied. Despite this, precise mechanisms of the key O─O cleavage step remain partly elusive to date; effects observed in various enzyme mutants remain partly unexplained. We have carried out extended (to 1000 ns) MM-MD and follow-on quantum mechanics/molecular mechanics computations, both on the well-studied FeOO state and on Cpd(0) (compound 0). Our simulations include (all camphor-bound): (a) WT (wild type), FeOO state. (b) WT, Cpd(0). (c) Pdx (Putidaredoxin, redox partner of P450)-docked-WT, FeOO state. (d) Pdx-docked WT, Cpd(0). (e) Pdx-docked T252A mutant, Cpd(0). Among our key findings: (a) Effect of Pdx docking appears to go far beyond that indicated in prior studies: it leads to specific alterations in secondary structure that create the crucial proton relay network. (b) Specific proton relay networks we identify are: FeOO(H)⋯T252⋯nH 2 O⋯D251 in WT; FeOO(H)⋯nH 2 O⋯D251 in T252A mutant; both occur with Pdx docking. (c) Direct interaction of D251 with -FeOOH is, respectively, rare/frequent in WT/T252A mutant. (d) In WT, T252 is in the proton relay network. (e) Positioning of camphor appears significant: when camphor is part of H-bonding network, second protonation appears to be facilitated.

An Intermediate Conformational State of Cytochrome P450cam-CN in Complex with Putidaredoxin.

Cytochrome P450cam is an archetypal example of the vast family of heme monooxygenases and serves as a model for an enzyme that is highly specific for both its substrate and reductase. During catalysis, it undergoes significant conformational changes of the F and G helices upon binding its substrate and redox partner, putidaredoxin (Pdx). Recent studies have shown that Pdx binding to the closed camphor-bound form of ferric P450cam results in its conversion to a fully open state. However, during catalytic turnover, it remains unclear whether this same conformational change also occurs or whether it is coupled to the formation of the critical compound I intermediate. Here, we have examined P450cam bound simultaneously by camphor, CN-, and Pdx as a mimic of the catalytically competent ferrous oxy-P450cam-Pdx state. The combined use of double electron-electron resonance and molecular dynamics showed direct observation of intermediate conformational states of the enzyme upon CN- and subsequent Pdx binding. This state is coupled to the movement of the I helix and residues at the active site, including Arg-186, Asp-251, and Thr-252. These movements enable occupation of a water molecule that has been implicated in proton delivery and peroxy bond cleavage to give compound I. These findings provide a detailed understanding of how the Pdx-induced conformational change may sequentially promote compound I formation followed by product release, while retaining stereoselective hydroxylation of the substrate of this highly specific monooxygenase.

Cytochrome P450-The Wonderful Nanomachine Revealed through Dynamic Simulations of the Catalytic Cycle.

This Account addresses the catalytic cycle of the enzyme cytochrome P450 (CYP450) as a prototypical biological machine with automatic features. CYP450 is a nanomachine that uses dioxygen and two reducing and two proton equivalents to oxidize a plethora of molecules (so-called substrates) as a means of supplying bio-organisms with essential molecules (e.g., brain neurotransmitters, sex hormones, etc.) and protecting biosystems against poisoning. An enticing property of CYP450s is that entrance of an oxidizable substrate into the active site initiates a series of events that constitute the catalytic cycle, which functions "automatically" in a regulated sequence of events culminating in the production of the oxidized substrates (e.g., hydroxylated, epoxidized, etc.), oftentimes with remarkable stereo- and regioselectivities. It is timely to demonstrate how theory uses molecular dynamics (MD) simulations and quantum-mechanical/molecular-mechanical (QM/MM) calculations to complement experiments and elucidate the choreography by which the protein regulates the catalytic cycle. CYP450 is a heme enzyme that contains a ferric ion (FeIII) coordinated by a porphyrin ligand, a water molecule, and a cysteinate ligand that is provided by a strategic residue of the encapsulating protein. While many of the individual steps are sufficiently well-understood, we shall provide here an overview of the factors that cause all of the steps to be sequentially coordinated. To this end, we use examples from three different CYP450 enzymes: the bacterial ones CYP450BM3 and CYP450CAM and the mammalian enzyme CYP4503A4. The treatment is limited to the catalytic cycle, as aspects of two-state reactivity were reviewed previously (e.g., Shaik , S. ; et al. Chem. Rev. 2005 , 105 , 2279 ). What are the principles that govern the seeming automatic feature? For example, how do substrate entrance and binding gate the enzyme? How does the reductase attachment to the enzyme affect the next steps? What triggers the attachment of the reductase? How does the electron transfer (ET) that converts FeIII to FeII occur? Is the ET coordinated with the entrance of O2 into the active site? What is the mechanism of the latter step? Since the entrance of the substrate expels the water molecules from the active site, how do water molecules re-enter to form a proton channel, which is necessary for creating the ultimate oxidant Compound I? How do mutations that disrupt the water channel nevertheless create a competent oxidant? By what means does the enzyme produce regio- and stereoselective oxidation products? What triggers the departure of the oxidized product, and how does the exit occur in a manner that generates the resting state ready for the next cycle? This Account shows that the entrance of the substrate triggers all of the ensuing events.