The cytochrome P450s are a superfamily of enzymes that are found in all kingdoms of living organisms, and typically catalyze the oxidative addition of atomic oxygen to an unactivated C-C or C-H bond. Over 8000 nonredundant sequences of putative and confirmed P450 enzymes have been identified, but three-dimensional structures have been determined for only a small fraction of these. While all P450 enzymes for which structures have been determined share a common global fold, the flexibility and modularity of structure around the active site account for the ability of P450 enzymes to accommodate a vast number of structurally dissimilar substrates and support a wide range of selective oxidations. In this review, known P450 structures are compared, and some structural criteria for prediction of substrate selectivity and reaction type are suggested. The importance of dynamic processes such as redox-dependent and effector-induced conformational changes in determining catalytic competence and regio-and stereoselectivity is discussed, and noncrystallographic methods for characterizing P450 structures and dynamics, in particular, mass spectrometry and nuclear magnetic resonance spectroscopy are reviewed. Antioxid. Redox Signal. 13, 1273-1296.

Cytochrome P450 monooxygenases typically catalyze the insertion of one atom of oxygen from O2 into unactivated carbon-hydrogen and carbon-carbon bonds, with concomitant reduction of the other oxygen atom to H2O by NAD(P)H. Comparison of the average structures of the camphor hydroxylase cytochrome P450cam (CYP101) obtained from residual dipolar coupling (RDC)-restrained molecular dynamics (MD) in the presence and absence of substrate camphor shows structural displacements resulting from the essential collapse of the active site upon substrate removal. This collapse has conformational consequences that extend across the protein structure, none of which were observed in analogous crystallographic structures. Mutations were made to test the involvement of the observed conformational changes in substrate binding and recognition. All of the mutations performed based upon the NMR-detected perturbations, even those remote from the active site, resulted in modified substrate selectivity, enzyme efficiency and/or haem iron spin state. The results demonstrate that solution NMR can provide insights into enzyme structure-function relationships that are difficult to obtain by other methods.

The two-protein complex between putidaredoxin (Pdx) and cytochrome P450(cam) (CYP101) is the catalytically competent species for camphor hydroxylation by CYP101. We detected a conformational change in CYP101 upon binding of Pdx that reorients bound camphor appropriately for hydroxylation. Experimental evidence shows that binding of Pdx converts a single X-proline amide bond in CYP101 from trans or distorted trans to cis. Mutation of proline 89 to isoleucine yields a mixture of both bound camphor orientations, that seen in Pdx-free and that seen in Pdx-bound CYP101. A mutation in CYP101 that destabilizes the cis conformer of the Ile 88-Pro 89 amide bond results in weaker binding of Pdx. This work provides direct experimental evidence for involvement of X-proline isomerization in enzyme function.

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The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities towards their common substrates (1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M +2 metal ion bound in the active site. The Ni +2 -bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate and carbon monoxide, while the Fe +2 -bound FeARD' catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS) and isothermal calorimetry measurements, it is concluded that the same four residues, His 96, His 98, Glu 102 and His 140, provide the protein-based ligands for the metal in both the Ni-and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD' and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a His ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved non-ligand acidic residues, Glu 95 and Glu 100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from in vivo sources or prevent the loss of metal to stronger chelators. The Glu 100 mutant reconstitutes readily but has low activity. Mutation of Asp 101 results in an active enzyme that incorporates metal in vivo but shows evidence of mixed forms.* To whom correspondence should be addressed: pochapsk@brandeis.edu, phone 781−736−2559, fax 781−736−2516. e Current address: Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, 70 Ship Street, GE3, Providence, RI, 02912 f Current address: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., Room 1007, Indianapolis IN 46202 g This work was supported in part by USPHS grant (R01-GM067786 TCP). Acknowledgment is made to the donors of The American Chemical Society Petroleum Research Fund for partial support (MJM). Trainee funding (SCC) was provided by the NIH-Chemistry Biology Interface Program, T32-GM08515. XAS data collection at the National Synchrotron Light Source at Brookhaven National Laboratory was supported by the U.S. Department of Energy, Division of Materials Sciences and Division of Chemical Sciences. Beamline X9B at NSLS is supported in part by the NIH.Supporting information available Fits for resting state and ES...

Residual dipolar couplings were used as restraints in fully solvated molecular dynamics simulations of reduced substrate-and carbonmonoxy-bound cytochrome P450 cam (CYP101A1), a 414-residue soluble monomeric heme-containing camphor monooxygenase from the soil bacterium Pseudomonas putida. The 1 D NH residual dipolar couplings used as restraints were measured in two independent alignment media. A soft annealing protocol was used to heat the starting structures while incorporating the RDC restraints. After production dynamics, structures with the lowest total violation energies for RDC restraints were extracted to identify ensembles of conformers accessible to the enzyme in solution. The simulations result in substrate orientations different from that seen in crystallographic structures and a more open and accessible enzyme active site, and largely support previously reported differences between the open and closed states of CYP101A1.The sine qua non of enzyme function is that the three-dimensional structure of an enzyme stabilizes the transition state of the reaction being catalyzed (1). This implies that the enzyme structure cannot stabilize the reactant (or product) ground states, at least not at the point of catalysis. However, enzymes have another job to do as well: They must concentrate reactants in the active site, that is, the thermodynamics of substrate binding must be sufficiently favorable so that substrate is bound selectively and with reasonable affinity. How then to square this particular circle? One way is to assume an enzyme is able to access different conformations in order to accomplish both tasks (2). If this is true, one would expect to find accessible "open" enzyme conformers that allow substrate access and bind substrate with a favorable free energy, and a different set of "closed" conformers that stabilize the appropriate transition states for catalysis. Furthermore, the activation barriers separating the open and closed conformers should be sufficiently high that each a This work was supported in part by a grant from the USPHS (R01-GM44191, TCP). EKA and JDM acknowledge support from USPHS grant R01DA027806, a Department of Education grant P116Z090309 and NSF support through Teragrid (MCB060061N). b The REP structure discussed in this paper has been deposited as PDB entry 2l8m. NMR chemical shifts and RDC restraints are deposited as BMRB entry 17415. * Inquiries should be addressed to this author at Dept. of Chemistry, MS 015, Brandeis University, 415 South St., Waltham, MA 02454-9110. Tel.: 781-736-2559, fax: 781-736-2516, pochapsk@brandeis.edu, website: www.chem.brandeis.edu/pochapsky. Supplementary Material Available: PDB format structure file for REP structure, RDC restraints in AMBER format and two tables discussed in the text are available in online at http://pubs.acs.org. conformation is discretely accessible on the time scale of catalysis, but not so formidable as to preclude access to either. NIH Public AccessWe have used multidimensional nuclear magnetic resonance (NMR) sp...

Local protein backbone dynamics of the camphor hydroxylase cytochrome P450cam (CYP101) depend upon the oxidation and ligation state of the heme iron. 1H,15N correlation nuclear magnetic resonance experiments were used to compare backbone dynamics of oxidized and reduced forms of this 414-residue metalloenzyme via hydrogen-deuterium exchange kinetics (H/D exchange) and 15N relaxation measurements, and these results are compared with previously published results obtained by H/D exchange mass spectrometry. In general, the reduced enzyme shows lower amplitude motions of secondary structural features than the oxidized enzyme on all of the time scales accessible to these experiments, and these differences are more pronounced in regions of the enzyme involved in substrate access to the active site (B′ helix, β3 and β5 sheets) and binding of putidaredoxin (C and L helices) the iron-sulfur protein that acts as effector and reductant of CYP101 in vivo. These results are interpreted in terms of local structural effects of changes in the heme oxidation state, and relevance of the observed effects to the enzyme mechanism is discussed.

A hydrogen bond network has been identified that adjusts protein-substrate contacts in cytochrome P450 cam (CYP101A1). Replacing the native substrate camphor with adamantanone or norcamphor causes perturbations in NMR-detected NH correlations assigned to the network, which includes portions of aβ sheet and an adjacent helix that is remote from the active site. A mutation in this helix reduces enzyme efficiency and perturbs the extent of substrate-induced spin state changes at the haem iron that accompany substrate binding. In turn, the magnitude of the spin state changes induced by alternate substrate binding parallel the NMR-detected perturbations observed near the haem in the enzyme active site. Keywordsenzyme; haem; nuclear magnetic resonance; site-directed mutagenesis One of the distinguishing characteristics of enzymes is their ability to selectively bind a particular substrate. The origin of this selectivity remains a puzzle. Typically, a group of related enzymes (isozymes) with a common function exhibit a well-conserved fold, with little variation in tertiary structure. Furthermore, catalytically important residues in the active site are usually strongly conserved and mutation of these residues often renders the enzyme inactive. But if active site residues and overall tertiary structure are conserved, how is substrate selectivity and product specificity enforced? The answers must lie in the complex interactions between protein structure and dynamics that are only now being investigated.The issue of substrate recognition and selectivity is of particular interest for the superfamily of cytochrome P450 monooxygenases. These enzymes are found in all kingdoms of life, and oxidize a bewildering variety of substrates. While many P450s are non-specific (particularly those involved in xenobiotic metabolism in vertebrates), other members of the superfamily catalyze highly regio-and stereospecific reactions. Examples include P450 EryF and EryK, which catalyze specific macrolide ring oxidations in the biosynthetic pathway of erythromycin, 1, 2 sterol biosynthetic enzymes such as aromatase 3 and bacterial catabolic enzymes, including the well-known examples of cytochrome P450 cam 4 and P450 terp . 5 Despite the multiplicity of P450 enzyme-substrate combinations, the P450 fold is highly conserved, with identical folding topology for all of the enzymes examined to date. 6 Structurally characterized P450s also show remarkable consistency in secondary structure as * Correspondence should be addressed to this author, pochapsk@brandeis.edu, phone 011-781-736-2559, website www.chem.brandeis.edu/pochapsky. NIH Public Access Author ManuscriptMetallomics. Author manuscript; available in PMC 2012 April 1. 12 We demonstrated that the solution conformation adopted by CYP101A1 in the absence of the effector protein putidaredoxin differs significantly from crystallographic structures. [13][14][15] We now show that NMR methods can provide insight into enzyme-substrate interactions by identifying structural features that are ...