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Putidaredoxin (Pdx) is the exclusive reductase and a structural effector for P450cam (CYP101A1). However, the mechanism of how Pdx modulates the conformational states of P450cam remains elusive. Here we report a putative communication pathway for the Pdx-induced conformational change in P450cam using results of double electron−electron resonance (DEER) spectroscopy and molecular dynamics simulations. Use of solution state DEER measurements allows us to observe subtle conformational changes in the internal helices in P450cam among closed, open, and P450cam−Pdx complex states. Molecular dynamics simulations and dynamic network analysis suggest that Pdx binding is coupled to small coordinated movements of several regions of P450cam, including helices C, B′, I, G, and F. These changes provide a linkage between the Pdx binding site on the proximal side of the enzyme and helices F/G on the distal side and the site of the largest movement resulting from the Pdx-induced closed-to-open transition. This study provides a detailed rationale for how Pdx exerts its long-recognized effector function at the active site from its binding site on the opposite face of the enzyme.

CYP119, a bacterial thermophilic protein from the cytochrome P450 superfamily, has previously been observed in three different conformations with different inhibitors bound using X-ray crystallography. The significance of these states in solution and in the function of the enzyme is not well-known. Double electron-electron resonance (DEER) was used to measure distances and distance distributions between spin-labels for populated conformational states in solution. DEER spectroscopy and molecular dynamics for the ligand-free enzyme suggest that the G helix is in a slightly different conformation than seen previously by crystallography, with the F/G loop in a slightly open conformation. Inhibitor-bound samples showed that this conformation remains as the predominant form, but partial conversion is indicated to a more closed conformation of the F/G loop. However, when the enzyme binds to lauric acid, the proposed substrate, it induces the conversion to a state that is characterized by increased disorder. We propose that similar to recent results with soluble CYP3A4, binding of the inhibitor to CYP119 is accompanied by only small changes in the enzyme structure, but substrate binding results in greater heterogeneity in the structure of the F/G loop region.

Various biophysical methods have provided evidence of a second substrate binding site in the well-studied cytochrome P450cam, although the location and biological relevance of this site has remained elusive. A related question is how substrate and product binding and egress occurs. While many active site access channels have been hypothesized, only one, channel 1, has been experimentally validated. In this study, molecular dynamics simulations reveal an allosteric site related to substrate binding and product egress. The remote allosteric site opens channel 1 and primes the formation of a new channel that is roughly perpendicular to channel 1. Substrate entry to the active site via channel 1 as well as substrate/product egress via channel 2 is observed after binding of a second molecule of substrate to the allosteric site, indicating cooperativity between these two sites. These results are consistent with and bring together many early and recent experimental results to reveal a dynamic interplay between a weak allosteric site and substrate binding to the active site that controls P450cam activity.

Manganese-oxide minerals (MnOx) are widely distributed over the Earth's surface, and their geochemical cycling is globally important. A multicopper oxidase (MCO) MnxG protein from marine Bacillus bacteria plays an essential role in producing MnOx minerals by oxidizing Mn2+(aq) at rates that are 3 to 5 orders of magnitude faster than abiotic rates. The MnxG protein is isolated as part of a multiprotein complex denoted as "Mnx" that includes accessory protein subunits MnxE and MnxF, with an estimated stoichiometry of MnxE3F3G and corresponding molecular weight of ≈211 kDa. Herein, we report successful expression and isolation of the MCO MnxG protein without the E3F3 hexamer. This isolated MnxG shows activity for Mn2+(aq) oxidation to form manganese oxides. The complement of paramagnetic Cu(II) ions in the Mnx protein complex was examined by electron paramagnetic resonance (EPR) spectroscopy. Two distinct classes of type 2 Cu sites were detected. One class of Cu(II) site (denoted as T2Cu-A), located in the MnxG subunit, is identified by the magnetic parameters g∥ = 2.320 and A∥ = 510 MHz. The other class of Cu(II) sites (denoted as T2Cu-B) is characterized by g∥ = 2.210 and A∥ = 615 MHz and resides in the putative hexameric MnxE3F3 subunit. These different magnetic properties correlate with the differences in the reduction potentials of the respective Cu(II) centers. These studies provide new insights into the molecular mechanism of manganese biomineralization.

Human cytochrome P450 3A4 (CYP3A4) is a membrane-associated monooxygenase that is responsible for metabolizing >50% of the pharmaceuticals in the current market, so studying its chemical mechanism and structural changes upon ligand binding will help provide deeper insights into drug metabolism and further drug development. The best-characterized cytochrome P450 is a bacterial form, P450cam, which undergoes significant conformational changes upon binding substrate and its redox partner, putidaredoxin. In contrast, most crystal structures of CYP3A4 with or without ligands have shown few changes, although allosteric effects and multiple-substrate binding in solution are well-documented. In this study, we use double electron–electron resonance (DEER) to measure distances between spatially separated spin-labels on CYP3A4 and molecular dynamics to interpret the DEER data. These methods were applied to a soluble N-terminally truncated CYP3A4 form, and the results show that there are few changes in the average structure upon binding ketoconazole, ritonavir, or midazolam. However, binding of midazolam, but not ketoconazole or ritonavir, resulted in a significant change in the motion and/or disorder in the F/G helix region near the substrate binding pocket. These results suggest that soluble CYP3A4 behaves in a unique way in response to inhibitor and substrate binding.

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.

Background: In order to perform selective C-H functionalization upon visible light irradiation, Ru(II)-diimine functionalized P450 heme enzymes have been developed. The sL407C-1 enzyme containing the Ru(bpy)_2PhenA (bpy = 2,2′-bipyridine and PhenA = 5-acetamido-1,10-phenanthroline) photosensitizer (1) covalently attached to the non-native single cysteine L407C of the P450BM3 heme domain mutant, displays high photocatalytic activity in the selective C-H bond hydroxylation of several substrates. Methods: A combination of X-ray crystallography, site-directed mutagenesis, transient absorption measurements and enzymatic assays was used to gain insights into its photocatalytic activity and electron transfer pathway. Results: The crystal structure of the sL407C-1 enzyme was solved in the open and closed conformations revealing a through-space electron transfer pathway involving highly conserved, F393 and Q403, residues. Several mutations of these residues (F393A, F393W or Q403W) were introduced to probe their roles in the overall reaction. Transient absorption measurements confirm rapid electron transfer as heme reduction is observed in all four hybrid enzymes. Compared to the parent sL407C-1, photocatalytic activity was negligible in the dF393A-1 enzyme while 60% increase in activity with total turnover numbers of 420 and 90% product conversion was observed with the dQ403W-1 mutant. Conclusions: In the sL407C-1 enzyme, the photosensitizer is ideally located to rapidly deliver electrons, using the naturally occurring electron transfer pathway, to the heme center in order to activate molecular dioxygen and sustain photocatalytic activity. General significance: The results shed light on the design of efficient light-driven biocatalysts and the approach can be generalized to other members of the P450 superfamily.

The cytochromes P450 form an enormous family of over 20 000 enzyme variants found in all branches of life. They catalyze the O2 dependent monooxygenation of a wide range of substrates in reactions important to drug metabolism, biosynthesis and energy utilization. Understanding how they function is important for biomedical science and requires a full description of their notorious propensity for specificity and promiscuity. The bacterial P450cam is an unusual example, having the most well characterized chemical mechanism of all of the forms. It also undergoes an increasingly well characterized structural change upon substrate binding, which may be similar to to that displayed by some, but not all forms of P450. Finally, P450cam is one of the rare forms that have a strict requirement for a particular electron donor, putidaredoxin (pdx). Pdx provides the required electrons for enzyme turnover, but it also induces specific changes in the enzyme to allow enzyme turnover, long known as its effector role. This review summarizes recent crystallographic and double electron–electron resonance studies that have revealed the effects of substrate and pdx binding on the structure of P450cam. We describe an emerging idea for how pdx exerts its effector function by inducing a conformational change in the enzyme. This change then propagates to the active site to enable cleavage of the ferric–hydroperoxy bond during catalysis, and appears to provide a very elegant approach for P450cam to attain both high efficiency and protection from oxidative damage.

Photo-induced charge-transfer dynamics and the influence of cluster size on the dynamics were investigated using five iron-sulfur clusters: the 1Fe-4S cluster in Pyrococcus furiosus rubredoxin, the 2Fe-2S cluster in Pseudomonas putida putidaredoxin, the 4Fe-4S cluster in nitrogenase iron protein, and the 8Fe-7S P-cluster and the 7Fe-9S-1Mo FeMo cofactor in nitrogenase MoFe protein. Laser excitation promotes the iron-sulfur clusters to excited electronic states that relax to lower states. The electronic relaxation lifetimes of the 1Fe-4S, the 8Fe-7S, and the 7Fe-9S-1Mo clusters are in the picoseconds timescale, although the dynamics of the MoFe protein is a mixture of the dynamics of the later two clusters. The lifetimes of the 2Fe-2S and the 4Fe-4S clusters, however, extend to several nanoseconds. A competition between reorganization energies and density of electronic states (thus electronic coupling between states) mediates the charge-transfer lifetimes, with the 2Fe-2S cluster of Pdx and the 4Fe-4S cluster of Fe protein lying at the optimum leading to them having significantly longer lifetimes. Their long lifetimes make them the optimal candidates for long-range electron transfer and as external photosensitizers for other photo-activated chemical reactions like solar hydrogen production. Potential electron-transfer and hole-transfer pathways are proposed that possibly facilitate these charge transfers.

Double electron-electron resonance (DEER) spectroscopy was used to determine the conformational state in solution for the heme monooxygenase P450cam when bound to its natural redox partner, putidaredoxin (Pdx). When oxidized Pdx was titrated into substrate-bound ferric P450cam, the enzyme shifted from the closed to the open conformation. In sharp contrast, however, the enzyme remained in the closed conformation when ferrous-CO P450cam was titrated with reduced Pdx. This result fully supports the proposal that binding of oxidized Pdx to P450cam opposes the open-to-closed transition induced by substrate binding. However, the data strongly suggest that in solution, binding of reduced Pdx to P450cam does not favor the open conformation. This supports a model in which substrate recognition is associated with the open-to-closed transition and electron transfer from Pdx occurs in the closed conformation. The opening of the enzyme in the ferric-hydroperoxo state following electron transfer from Pdx would provide for efficient O2 bond activation, substrate oxidation, and product release.

In this study, the effector role of Pdx (putidaredoxin) on cytochrome P450cam conformation is refined by attaching two different spin labels, MTSL or BSL (bifunctional spin-label) onto the F or G helices and using DEER (double electron-electron resonance) to measure the distance between labels. Recent EPR and crystallographic studies have observed that oxidized Pdx induces substrate-bound P450cam to change from the closed to the open state. However, this change was not observed by DEER in the reduced Pdx complex with carbon-monoxide-bound P450cam (Fe(2+)CO). In addition, recent NMR studies have failed to observe a change in P450cam conformation upon binding Pdx. Hence, resolving these issues is important for a full understanding the effector role of Pdx. Here we show that oxidized Pdx induces camphor-bound P450cam to shift from the closed to the open conformation when labeled on either the F or G helices with MTSL. BSL at these sites can either narrow the distance distribution widths dramatically or alter the extent of the conformational change. In addition, we report DEER spectra on a mixed oxidation state containing oxidized Pdx and ferrous CO-bound P450cam, showing that P450cam remains closed. This indicates that CO binding to the heme prevents P450cam from opening, overriding the influence exerted by Pdx binding. Finally, we report the open form P450cam crystal structure with substrate bound, which suggests that crystal packing effects may prevent conformational conversion. Using multiple labeling approaches, DEER provides a unique perspective to resolve how the conformation of P450cam depends on Pdx and ligand states.

Conformational changes in the substrate access channel have been observed for several forms of cytochrome P450, but the extent of conformational plasticity exhibited by a given isozyme has not been completely characterized. Here we present crystal structures of P450cam bound to a library of 12 active site probes containing a substrate analog tethered to a variable linker. The structures provide a unique view of the range of protein conformations accessible during substrate binding. Principal component analysis of a total of 30 structures reveals three discrete clusters of conformations; closed (P450cam-C), intermediate (P450cam-I) and fully open (P450cam-O). Relative to P450cam-C, the P450cam-I state results predominantly from a retraction of the F-helix, while both F and G helices move in concert to reach the fully open P450cam-O state. Both P450cam-C and P450cam-I are well defined states, while P450cam-O shows evidence for a somewhat broader distribution of conformations, and includes the open form recently seen in the absence of substrate. The observed clustering of protein conformations over a wide range of ligand variants suggests a multi-step closure of the enzyme around the substrate that begins by conformational selection from an ensemble of open conformations and proceeds through a well defined intermediate, P450cam-I, before full closure to the P450cam-C state in the presence of small substrates. This multi-step pathway may have significant implications for a full understanding of substrate specificity, kinetics and coupling of substrate binding to P450 function.

P450cam from Pseudomonas putida is the best characterized member of the vast family of cytochrome P450s, and it has long been believed to have a more rigid and closed active site relative to other P450s. Here we report X-ray structures of P450cam crystallized in the absence of substrate and at high and low [K(+)]. The camphor-free structures are observed in a distinct open conformation characterized by a water-filled channel created by the retraction of the F and G helices, disorder of the B' helix, and loss of the K(+) binding site. Crystallization in the presence of K(+) alone does not alter the open conformation, while crystallization with camphor alone is sufficient for closure of the channel. Soaking crystals of the open conformation in excess camphor does not promote camphor binding or closure, suggesting resistance to conformational change by the crystal lattice. This open conformation is remarkably similar to that seen upon binding large tethered substrates, showing that it is not the result of a perturbation by the ligand. Redissolved crystals of the open conformation are observed as a mixture of P420 and P450 forms, which is converted to the P450 form upon addition of camphor and K(+). These data reveal that P450cam can dynamically visit an open conformation that allows access to the deeply buried active site without being induced by substrate or ligand.

To evaluate the potential of using heme-containing lipocalin nitrophorin 1 (NP1) as a template for protein engineering, we have replaced the native axial heme-coordinating histidine residue with glycine, alanine, and cysteine. We report here the characterization of the cysteine mutant H60C_NP1 by spectroscopic and crystallographic methods. The UV/vis, resonance Raman, and magnetic circular dichroism spectra suggest weak thiolate coordination of the ferric heme in the H60C_NP1 mutant. Reduction to the ferrous state resulted in loss of cysteine coordination, while addition of exogenous imidazole ligands gave coordination changes that varied with the ligand. Depending on the substitution of the imidazole, we could distinguish three heme coordination states: five-coordinate monoimidazole, six-coordinate bisimidazole, and six-coordinate imidazole/thiolate. Ligand binding affinities were measured and found to be generally 2-3 orders of magnitude lower for the H60C mutant relative to NP1. Two crystal structures of the H60C_NP1 in complex with imidazole and histamine were solved to 1.7- and 1.96-A resolution, respectively. Both structures show that the H60C mutation is well tolerated by the protein scaffold and suggest that heme-thiolate coordination in H60C_NP1 requires some movement of the heme within its binding cavity. This adjustment may be responsible for the ease with which the engineered heme-thiolate coordination can be displaced by exogenous ligands.

Nitric oxide synthase (NOS) is the primary biological source of the ubiquitous signaling molecule, nitric oxide (•NO). The enzyme utilizes tetrahydrobiopterin (H4B) as an essential cofactor, where it plays a key role in the catalytic conversion of L-arginine to citrulline and •NO.1 The pterin has been shown to serve both structural and catalytic roles in the enzyme by affecting the monomer to dimer transition, promoting protein stability, and forming a radical cation during catalytic turnover.2

Members of the ubiquitous cytochrome P450 family catalyze a vast range of biologically significant reactions in mammals, plants, fungi, and bacteria. Some P450s display a remarkable promiscuity in substrate recognition, while others are very specific with respect to substrate binding or regio and stereo-selective catalysis. Recent results have suggested that conformational flexibility in the substrate access channel of many P450s may play an important role in controlling these effects. Here, we report the X-ray crystal structures at 1.8A and 1.5A of cytochrome P450cam complexed with two synthetic molecular wires, D-4-Ad and D-8-Ad, consisting of a dansyl fluorophore linked to an adamantyl substrate analog via an alpha,omega-diaminoalkane chain of varying length. Both wires bind with the adamantyl moiety in similar positions at the camphor-binding site. However, each wire induces a distinct conformational response in the protein that differs from the camphor-bound structure. The changes involve significant movements of the F, G, and I helices, allowing the substrate access channel to adapt to the variable length of the probe. Wire-induced opening of the substrate channel also alters the I helix bulge and Thr252 at the active site with binding of water that has been proposed to assist in peroxy bond cleavage. The structures suggest that the coupling of substrate-induced conformational changes to active-site residues may be different in P450cam and recently described mammalian P450 structures. The wire-induced changes may be representative of the conformational intermediates that must exist transiently during substrate entry and product egress, providing a view of how substrates enter the deeply buried active site. They also support observed examples of conformational plasticity that are believed be responsible for the promiscuity of drug metabolizing P450s. Observation of such large changes in P450cam suggests that substrate channel plasticity is a general property inherent to all P450 structures.

Nitric oxide synthases (NOS) are a family of cysteine thiolate-ligated heme-containing monooxygenases that catalyze the NADPH-dependent two-step conversion of L-arginine to NO and L-citrulline. During the catalysis, a portion of the NOS heme forms an inhibitory complex with self-generated NO that is subsequently reverted back to NO-free active enzyme under aerobic conditions, suggesting a downstream regulator role of NO. Recent studies revealed that mutation of a conserved proximal tryptophan-409, which forms one of three hydrogen bonds to the heme-coordinated cysteine thiolate, to tyrosine or phenylalanine considerably increases the turnover number of neuronal NOS (nNOS). To further understand these properties of nNOS on its active site structural level, we have examined the oxygenase (heme-containing) domain of the two mutants in close comparison with that of wild-type nNOS with UV-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy. Among several oxidation and ligation states examined, only the ferrous-NO adducts of the two mutants exhibit spectra that are markedly distinct from those of parallel derivatives of the wild-type protein. The spectra of the ferrous-NO mutants are broadly similar to those of known five-coordinate ferrous-NO heme complexes, suggesting that these mutants are predominantly five coordinate in their ferrous-NO states. The present results are indicative of cleavage of the Fe-S bond in the nNOS mutants in their ferrous-NO state and imply a significant role of the conserved tryptophan in stabilization of the Fe-S bond.

Electronic absorption and magnetic circular dichroism (MCD) data are reported for the H175G cavity mutant of yeast cytochrome c peroxidase (H175G CCP) in its ligand-free ferric state at 4 °C under slightly acidic conditions (pH 5.9). Initial analysis of the MCD spectrum of this state suggested a five-coordinate structure with ligation by an aspartate or glutamate. However, examination of the crystal structure of wild-type CCP revealed no plausible carboxylate-containing amino-acid residues close enough to the heme iron to serve as the ligand. An alternative interpretation of the MCD data is that the phosphate ion from the buffer is bound to the ferric heme iron. A phosphate group is similar in electronic character to a carboxylate and could give an MCD signal similar to that of a carboxylate-bound heme. Phosphate coordination to the ferric heme iron, albeit trans to imidazole, has recently been seen in a 2.0-A-resolution crystal structure of imidazole-bound ferric H175G CCP in phosphate buffer [Biochemistry 40 (5) (2001) 1265]. To investigate the validity of phosphate ligation, the pH 5.9 species in the 100 mM phosphate buffer was exchanged into the pH 5.9, 100 mM MES (2-[N-morpholino]ethanesulfonic acid) buffer. The MCD spectrum of the species in the MES buffer was spectrally distinct from that in the phosphate buffer, indicating that the original spectrum depends on the presence of a phosphate ion. We conclude that in the phosphate buffer, the exogenous ligand-free ferric state of the H175G CCP cavity mutant is actually coordinated by a phosphate ion from the buffer.

A previously proposed electron transfer (ET) pathway in the heme enzyme cytochrome c peroxidase has been excised from the structure, leaving an open ligand-binding channel in its place. Earlier studies on cavity mutants of this enzyme have revealed structural plasticity in this region of the molecule. Analysis of these structures has allowed the design of a variant in which the specific section of protein backbone representing a previously proposed ET pathway is accurately extracted from the protein. A crystal structure verified the creation of an open channel that overlays the removed segment, extending from the surface of the protein to the heme at the core of the protein. A number of heterocyclic cations were found to bind to the proximal-channel mutant with affinities that can be rationalized based on the structures. It is proposed that small ligands bind more weakly to the proximal-channel mutant than to the W191G cavity due to an increased off rate of the open channel, whereas larger ligands are able to bind to the channel mutant without inducing large conformational changes. The structure of benzimidazole bound to the proximal-channel mutant shows that the ligand accurately overlays the position of the tryptophan radical center that was removed from the wild-type enzyme and displaces four of the eight ordered solvent molecules seen in the empty cavity. Ligand binding also caused a small rearrangement of the redesigned protein loop, perhaps as a result of improved electrostatic interactions with the ligand. The engineered channel offers the potential for introducing synthetic replacements for the removed structure, such as sensitizer-linked substrates. These installed "molecular wires" could be used to rapidly initiate reactions, trap reactive intermediates, or answer unresolved questions about ET pathways.

Cavity complementation has been observed in many proteins, where an appropriate small molecule binds to a cavity-forming mutant. Here, the binding of compounds to the W191G cavity mutant of cytochrome c peroxidase is characterized by X-ray crystallography and binding thermodynamics. Unlike cavities created by removal of hydrophobic side-chains, the W191G cavity does not bind neutral or hydrophobic compounds, but displays a strong specificity for heterocyclic cations, consistent with the role of the protein to stabilize a tryptophan radical at this site. Ligand dissociation constants for the protonated cationic state ranged from 6 μM for 2-amino-5-methylthiazole to 1 mM for neutral ligands, and binding was associated with a large enthalpy-entropy compensation. X-ray structures show that each of 18 compounds with binding behavior bind specifically within the artificial cavity and not elsewhere in the protein. The compounds make multiple hydrogen bonds to the cavity walls using a subset of the interactions seen between the protein and solvent in the absence of ligand. For all ligands, every atom that is capable of making a hydrogen bond does so with either protein or solvent. The most often seen interaction is to Asp235, and most compounds bind with a specific orientation that is defined by their ability to interact with this residue. Four of the ligands do not have conventional hydrogen bonding atoms, but were nevertheless observed to orient their most polar CH bond towards Asp235. Two of the larger ligands induce disorder in a surface loop between Pro190 and Asn195 that has been identified as a mobile gate to cavity access. Despite the predominance of hydrogen bonding and electrostatic interactions, the small variation in observed binding free energies were not correlated readily with the strength, type or number of hydrogen bonds or with calculated electrostatic energies alone. Thus, as with naturally occurring binding sites, affinities to W191G are likely to be due to a subtle balance of polar, non-polar, and solvation terms. These studies demonstrate how cavity complementation and judicious choice of site can be used to produce a protein template with an unusual ligand-binding specificity.

We report theoretical calculations of vibrational absorption and Raman spectra for the tryptophan analog 3-methylindole using density functional theory, the Becke3LYP hybrid functional, and the TZ/2P basis set. These results are compared to experimentally measured vibrational absorption and Raman spectra for 3-methylindole. The theoretical calculations represent accurate predictions of the observed vibrational frequencies and intensities, and of the Raman intensities. Currently, tryptophan radicals, either neutral or cationic, are believed to participate in electron transfer in enzymes such as cytochrome c peroxidase, ribonucleotide reductase, and DNA photolyase. In this paper we also report theoretical vibrational absorption and Raman spectra for 3-methylindole cation radical and 3-methylindole neutral radical. These predictions should provide specific spectroscopic markers for the detection of neutral or cationic tryptophan radicals in biological systems, providing a complement to the data available from electron paramagnetic resonance experiments. Raman spectroscopy of tryptophan is already in use in the study of protein conformations; theoretical predictions for the radical species provides a new tool for the detection of neutral or cation radicals of tryptophan in natural systems predisposed to the appropriate experiment.

Electronic absorption and magnetic circular dichroism (MCD) spectroscopic data at 4°C are reported for exogenous ligand-free ferric forms of cytochrome c peroxidase (CCP) in comparison with two other histidine-ligated heme proteins, horseradish peroxidase (HRP) and myoglobin (Mb). In particular, we have examined the ferric states of yeast wild-type CCP (YCCP), CCP(MKT) which is the form of the enzyme that is expressed in and purified from E. coli, and contains Met–Lys–Thr (MKT) at the N-terminus, CCP(MKT) in the presence of 60% glycerol, lyophilized YCCP, and alkaline CCP(MKT). The present study demonstrates that, while having similar electronic absorption spectra, the MCD spectra of ligand-free ferric YCCP and CCP(MKT) are somewhat varied from one another. Detailed spectral analyses reveal that the ferric form of YCCP, characterized by a long wavelength charge transfer (CT) band at 645 nm, exists in a predominantly penta-coordinate state with spectral features similar to those of native ferric HRP rather than ferric Mb (His/water hexa-coordinate). The electronic absorption spectrum of ferric CCP(MKT) is similar to those of the penta-coordinate states of ferric YCCP and ferric HRP including a CT band at 645 nm. However, its MCD spectrum shows a small trough at 583 nm that is absent in the analogous spectra of YCCP and HRP. Instead, this trough is similar to that seen for ferric myoglobin at about 585 nm, and is attributed (following spectral simulations) to a minor contribution (≤5%) in the spectrum of CCP(MKT) from a hexa-coordinate low-spin species in the form of a hydroxide-ligated heme. The MCD data indicate that the lyophilized sample of ferric YCCP (λCT=637 nm) contains considerably increased amounts of hexa-coordinate low-spin species including both His/hydroxide and bis-His species. The crystal structure of a spectroscopically similar sample of CCP(MKT) (λCT=637 nm) solved at 2.0 A resolution is consistent with His/hydroxide coordination. Alkaline CCP (pH 9.7) is proposed to exist as a mixture of hexa-coordinate, predominantly low-spin complexes with distal His 52 and hydroxide acting as distal ligands based on MCD spectral comparisons.

The addition of exogenous ligands to the ferric and ferrous states of yeast cytochrome c peroxidase (CCP) is investigated with magnetic circular dichroism (MCD) at 4°C to determine the effect the protein environment may exercise on spectral properties. The MCD spectrum of each derivative is directly compared to those of analogous forms of horseradish peroxidase (HRP) and myoglobin (Mb), two well-characterized histidine-ligated heme proteins. The ferric azide adduct of CCP is a hexacoordinate, largely low-spin species with an MCD spectrum very similar to that of ferric azide HRP. This complex displays an MCD spectrum dissimilar from that of the Mb derivative, possibly because of the stabilizing interaction between the azide ligand and the distal arginine of CCP (Arg 48). For the ferric fluoride derivative all three proteins display varied MCD data, indicating that the differences in the distal pocket of each protein influences their respective MCD characteristics. The MCD data for the cyanoferric complexes are similar for all three proteins, demonstrating that a strong field ligand bound in the sixth axial position dominates the MCD characteristics of the derivative. Similarly, the ferric NO complexes of the three proteins show MCD spectra similar in feature position and shape, but vary somewhat in intensity. Reduction of CCP at neutral pH yields a typical pentacoordinate high-spin complex with an MCD spectrum similar to that of deoxyferrous HRP. Formation of the NO and cyanide complexes of ferrous CCP gives derivatives with MCD spectra similar to the analogous forms of HRP and Mb in both feature position and shape. Addition of CO to deoxyferrous CCP results in a ferrous-CO complex with MCD spectral similarity to that of ferrous-CO HRP but not Mb, indicating that interactions between the ligand and the distal residues affects the MCD characteristics. Examination of alkaline (pH 9.7) deoxyferrous CCP indicates that a pH dependent conformational change has occurred, leading to a coordination structure similar to that of ferrous cytochrome b5, a known bis-histidine complex. Exposure of this complex to CO further confirms that a conformational change has taken place in that the MCD spectral characteristics of the resulting complex are similar to those of ferrous-CO Mb but not ferrous-CO HRP. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: S42–S52, 1999

Isotope labeling has been used to assign the resonance Raman spectra of cytochrome c peroxidase, expressed in Escherichia coli [CCP (MKT)], and of the D235N site mutant. 54Fe labeling establishes the coexistence of two separate bands (233 and 246 cm-1), arising from the stretching of the bond between the Fe atom and the proximal histidine ligand, His175. These are assigned to tautomers of the H-bond between the His175 imidazole NΓH proton and the Asp235 carboxylate side chain: In one tautomer the proton resides on the imidazole while in the other the proton is transferred to the carboxylate. When Asp235 is replaced by Asn, the H-bond is lost, and the Fe-His stretching frequency is markedly lowered. Two new RR bands are produced, at 205 and 185 cm-1, as a result of coupling between the shifted Fe-His vibration and a nearby porphyrin mode; the two bands share the 54Fe sensitivity expected for Fe-His stretching. C=C stretching and CβC=C bending vibrations have been separately assigned to the 2- and 4-vinyl groups of the protoheme prosthetic group via selective vinyl deuteration. In the acid form of the enzyme, the frequencies coincide for the two vinyl groups, at 1618 cm-1 for the C=C stretch, and at 406 cm-1 for the CβC=C bend. However, the 2-vinyl frequencies are elevated in the alkaline form of the enzyme, to 1628 cm-1 for C=C stretching, and to 418 cm-1 for CβC=C bending, while the 4-vinyl frequencies remain unshifted. Thus, the acid-alkaline transition involves a protein conformation change that specifically perturbs the 2-vinyl substituent. This perturbation might be a reorientation of the vinyl group, or an alteration of the porphyrin geometry that affects the porphyrin-vinyl coupling. The perturbation is attenuated when CO is bound to the enzyme; the C=C frequency is then unaffected in the alkaline form, while the CβC=C bending frequency is shifted to a smaller extent (412 cm-1). This attenuation is probably linked to inhibition of distal histidine binding to the heme Fe in the alkaline form when the CO is bound. © 1996 John Wiley & Sons, Inc.

The design of a series of functionally active models for manganese peroxidase (MnP) is described. Artificial metal binding sites were created near the heme of cytochrome c peroxidase (CCP) such that one of the heme propionates could serve as a metal ligand. At least two of these designs, MP6.1 and MP6.8, bind Mn2+ with Kd congruent with 0.2 mM, react with H2O2 to form stable ferryl heme species, and catalyze the steady-state oxidation of Mn2+ at enhanced rates relative to WT CCP. The kinetic parameters for this activity vary considerably in the presence of various dicarboxylic acid chelators, suggesting that the similar features displayed by native MnP are largely intrinsic to the manganese oxidation reaction rather than due to a specific interaction between the chelator and enzyme. Analysis of pre-steady-state data shows that electron transfer from Mn2+ to both the Trp-191 radical and the ferryl heme center of compound ES is enhanced by the metal site mutations, with transfer to the ferryl center showing the greatest stimulation. These properties are perplexingly similar to those reported for an alternate model for this site (1), despite rather distinct features of the two designs. Finally, we have determined the crystal structure at 1.9 A of one of our designs, MP6.8, in the presence of MnSO4. A weakly occupied metal at the designed site appears to coordinate two of the proposed ligands, Asp-45 and the heme 7-propionate. Paramagnetic nuclear magnetic resonance spectra also suggest that Mn2+ is interacting with the heme 7-propionate in MP6.8. The structure provides a basis for understanding the similar results of Yeung et al. (1), and suggests improvements for future designs.

We have measured hyperfine shifted NMR signals associated with deuterated imidazole bound to the high-spin Fe3+ state of cytochrome c peroxidase using deuterium magic angle spinning solid-state NMR. These experiments were performed on a mutant of cytochrome c peroxidase, CcP(H175G), for which replacement of the proximal histidine with glycine produced a cavity that can bind a variety of substituted imidazoles including imidazole or 2-methylimidazole. The mutant with imidazole bound is inactive; specifically its reaction with H2O2 is blocked. We observed deuterium NMR signals from the methyl-d3 group of the perdeuterated 2-MeIm sample bound to H175G CcP. The signal displayed an upfield chemical shift and exhibited non-Curie temperature dependence, indicating the existence of low-lying excited electronic states. Upon introducing a nondeuterated competitive ligand, imidazole, a decrease in the intensity of this signal was detected, consistent with the assignment of the deuterium signal to the bound 2-methyli...

The binding and oxidation of an artificial substrate, 2-aminothiazole, by an engineered cavity of cytochrome c peroxidase is described. The W191G mutant has been shown to create a buried cavity into which a number of small heterocyclic compounds will bind [Fitzgerald, M. M., Churchill, M. J., McRee, D. E., & Goodin, D. B. (1994) Biochemistry 33, 3807-3818], providing a specific site near the heme from which substrates might be oxidized. In this study, we show by titration calorimetry that 2-aminothiazole binds to W191G with a Kd of 0.028 mM at pH 6. A crystal structure at 2.3 A resolution of W191G in the presence of 2-aminothiazole reveals the occupation of this compound in the cavity, and indicates that it is in van der Waals contact with the heme. The WT enzyme reacts with H2O2 to form Compound ES, in which both the iron center and the Trp-191 side chain are reversibly oxidized. For the W191F (and perhaps the W191G) mutants, the iron is still oxidized, but the second equivalent exists transiently as a radical on the porphyrin before migrating to an alternate protein radical site [Erman, J. E., Vitello, L. B., Mauro, J. M., & Kraut, J. (1989) Biochemistry 28, 7992-7995]. Two separate reactions are observed between 2-aminothiazole and the oxidized centers of W191G. In the one reaction, optical and EPR spectra of the heme are used to show that 2-aminothiazole acts as an electron donor to the ferryl (Fe4+dO) center of W191G to reduce it to the ferric oxidation state. This reaction occurs from within the cavity, as it is not observed for variants that lack this artificial binding site. A second reaction between 2-aminothiazole and peroxide-oxidized W191G, which is much less efficient, results in the specific covalent modification of Tyr-236. Electrospray mass spectra of the W191G after incubation in 2-aminothiazole and H2O2 show a modification of the protein indicative of covalent binding of 2-aminothiazole. The site of modification was determined to be Tyr-236 by CNBr peptide mapping and automated peptide sequencing. The covalent modification is only observed for W191G and W191F which form the alternate radical center. This observation provides an unanticipated assignment of this free radical species to Tyr-236, which is consistent with previous proposals that it is a tyrosine. The oxidation of 2-aminothiazole by W191G represents an example of how the oxidative capacity inherent in the heme prosthetic group and the specific binding behavior of artificial protein cavities can be harnessed and redirected toward the oxidation of organic substrates.

Conformational changes that gate the access of substrates or ligands to an active site are important features of enzyme function. In this report, we describe an unusual example of a structural rearrangement near a buried artificial cavity in cytochrome c peroxidase that occurs on binding protonated benzimidazole. A hinged main-chain rotation at two residues (Pro 190 and Asn 195) results in a surface loop rearrangement that opens a large solvent-accessible channel for the entry of ligands to an otherwise inaccessible binding site. The trapping of this alternate conformational state provides a unique view of the extent to which protein dynamics can allow small molecule penetration into buried protein cavities.

Ab initio calculations have been carried out on 3-methylindole, and the cation and neutral radicals of 3-methylindole, using density functional theory (DFT), the Becke3−Lee−Yang−Parr functional, and the 6-31G*, 6-31+G*, 3-21G*, and TZ2P basis sets. Optimized geometries, vibrational frequencies, and for the radicals, atomic spin densites are calculated. The latter are compared to experimental spin densities recently determined for the tryptophan-191 radical of compound ES of the enzyme cytochrome-c-peroxidase. The DFT spin densities for the cation radical of 3-methylindole are in excellent agreement with the data for the tryptophan-191 radical, which supports the conclusion that the tryptophan radical is a cation radical. The results are compared to calculations using second-order Moller−Plesset theory (MP2) and the 6-31G** basis set. The MP2 spin densities are in significantly worse agreement with the experimental spin densities.

A key challenge in structure-based discovery is accounting for modulation of protein-ligand interactions by ordered and bulk solvent. To investigate this, we compared ligand binding to a buried cavity in Cytochrome c Peroxidase (CcP), where affinity is dominated by a single ionic interaction, versus a cavity variant partly opened to solvent by loop deletion. This opening had unexpected effects on ligand orientation, affinity, and ordered water structure. Some ligands lost over ten-fold in affinity and reoriented in the cavity, while others retained their geometries, formed new interactions with water networks, and improved affinity. To test our ability to discover new ligands against this opened site prospectively, a 534,000 fragment library was docked against the open cavity using two models of ligand solvation. Using an older solvation model that prioritized many neutral molecules, three such uncharged docking hits were tested, none of which was observed to bind; these molecules were not highly ranked by the new, context-dependent solvation score. Using this new method, another 15 highly-ranked molecules were tested for binding. In contrast to the previous result, 14 of these bound detectably, with affinities ranging from 8 µM to 2 mM. In crystal structures, four of these new ligands superposed well with the docking predictions but two did not, reflecting unanticipated interactions with newly ordered waters molecules. Comparing recognition between this open cavity and its buried analog begins to isolate the roles of ordered solvent in a system that lends itself readily to prospective testing and that may be broadly useful to the community.

The fully oxidized state of cytochrome c peroxidase (CcP), called ES, contains two oxidizing equivalents, one as an oxyferryl heme and the other as an organic radical on an amino acid residue. The unusual electron paramagnetic resonance spectrum of ES has been shown to be due to a weak distributed exchange coupling between the two paramagnetic redox centers. Various residues have been proposed as the radical site over the years. In this paper continuous wave and pulsed Q-band electron nuclear double resonance (ENDOR) spectroscopy confirms that the radical is located on Trp-191, as previously proposed. The paper completes the characterization of the active site of compound ES as being comprized of an oxyferryl heme coupled to the Trp-191 {pi}-cation radical by a weak spin exchange. 47 refs., 11 figs., 2 tabs.

The activated state of cytochrome c peroxidase, compound ES, contains a cation radical on the Trp-191 side chain. We recently reported that replacing this tryptophan with glycine creates a buried cavity at the active site that contains ordered solvent and that will specifically bind substituted imidazoles in their protonated cationic forms (Fitzgerald MM, Churchill MJ, McRee DE, Goodin DB, 1994, Biochemistry 33:3807-3818). Proposals that a nearby carboxylate, Asp-235, and competing monovalent cations should modulate the affinity of the W191G cavity for ligand binding are addressed in this study. Competitive binding titrations of the imidazolium ion to W191G as a function of [K+] show that potassium competes weakly with the binding of imidazoles. The dissociation constant observed for potassium binding (18 mM) is more than 3,000-fold higher than that for 1,2-dimethylimidazole (5.5 microM) in the absence of competing cations. Significantly, the W191G-D235N double mutant shows no evidence for binding imidazoles in their cationic or neutral forms, even though the structure of the cavity remains largely unperturbed by replacement of the carboxylate. Refined crystallographic B-values of solvent positions indicate that the weakly bound potassium in W191G is significantly depopulated in the double mutant. These results demonstrate that the buried negative charge of Asp-235 is an essential feature of the cation binding determinant and indicate that this carboxylate plays a critical role in stabilizing the formation of the Trp-191 radical cation.

A gene coding for the F172Y mutant of horseradish peroxidase isozyme C (HRP) has been constructed and expressed in both Spodoptera frugiperda (SF-9) and Trichoplusia ni egg cell homogenate (HighFive®) cells. Homology modeling with respect to three peroxidases for which crystal structures are available places Phe172 on the proximal side of the heme in the vicinity of porphyrin pyrrole ring C. The pH optimum and spectroscopic properties of the F172Y mutant are essentially identical to those of wild type HRP. Vmax values show that the mutant protein retains most of the guaiacol oxidizing activity. Stopped flow studies indicate that Compound I is formed with H2O2 at the same rate (k1 = 1.6 × 107M−1 s−1) at both pH 6.0 and 8.0 as it is with the wild type enzyme. This Compound I species decays rapidly at a rate k2 = 1.01 s−1, pH 7.0, to a second two-electron oxidized species that retains the ferryl (FeIV = O) absorption. EPR studies establish that a ferryl porphyrin radical cation is present in the initial Compound I, but electron transfer from the protein results in formation of a second Compound I species with an unpaired electron on the protein (presumably on Tyr172). The presence or absence of oxidizable amino acids adjacent to the heme is thus a key determinant of whether the second oxidation equivalent in Compound I is found as a porphyrin or protein radical cation.

Although cytochrome P450cam from Pseudomonas putida , the archetype for all heme monooxygenases, has long been known to have a closed active site, recent reports show that the enzyme can also be crystallized in at least two clusters of open conformations. This suggests that the enzyme may undergo significant conformational changes during substrate binding and catalytic turnover. However, these conformations were observed in the crystalline state, and information is needed about the conformations that are populated in solution. In this study, double electron–electron resonance experiments were performed to observe substrate-induced changes in distance as measured by the dipolar coupling between spin labels introduced onto the surface of the enzyme on opposite sides of the substrate access channel. The double electron–electron resonance data show a decrease of 0.8 nm in the distance between spin labels placed at S48C and S190C upon binding the substrate camphor. A rotamer distribution model based on the crystal structures adequately describes the observed distance distributions. These results demonstrate conclusively that, in the physiologically relevant solution state, the substrate-free enzyme exists in the open P450cam-O conformation and that camphor binding results in conversion to the closed P450cam-C form. This approach should be useful for investigating many other P450s, including mammalian forms, in which the role of conformational change is of central importance but not well understood.

The crystal structure of the His-175-->Gly (H175G) mutant of cytochrome-c peroxidase (EC 1.11.1.5), missing its only heme ligand, reveals that the histidine is replaced by solvent to give a bisaquo heme protein. This protein retains some residual activity, which can be stimulated or inhibited by addition of exogenous ligands. Structural analysis confirms the binding of imidazole to the heme at the position of the wild-type histidine ligand. This imidazole complex reacts readily with hydrogen peroxide to produce a radical species with novel properties. However, reactivation in this complex is incomplete (approximately 5%), which, in view of the very similar structures of the wild-type and the H175G/imidazole forms, implies a critical role for tethering of the axial ligand in catalysis. This study demonstrates the feasibility of constructing heme enzymes with no covalent link to the protein and with unnatural ligand replacements. Such enzymes may prove useful in studies of electron transfer mechanisms and in the engineering of novel heme-based catalysts.

In the oxidized "ES" state of cytochrome c peroxidase, Trp-191 is reversibly oxidized to a stable cation free radical by the hypervalent heme. To explore the potential for engineering a binding site for heterocyclic compounds at this site, the mutant W191G was constructed. Two independent crystal structures of W191G at 2.1- and 2.3-A resolution show that W191G contains a well-defined, approximately 180-A3 cavity at the Trp-191 site. The cavity is occupied by five ordered water molecules which participate in an extensive hydrogen-bonding network with each other, with polar main-chain atoms, and with the carboxylate of Asp-235. After a number of heterocyclic compounds were screened, evidence was obtained that substituted imidazoles bind to the cavity of W191G. Titration of W191G with imidazole resulted in a perturbation of the Soret absorption band that was not observed for W191H, W191F, or the native enzyme. The dissociation constants for binding of benzimidazole, imidazole, 2-ethylimidazole, 1-methylimidazole, 2-methylimidazole, and 1,2-dimethylimidazole to W191G were respectively 2.58, 0.70, 0.36, 0.057, 0.047, and 0.027 mM at pH 6.0. The highest binding affinity was exhibited by 1,2-dimethylimidazole, indicating that steric interactions and the efficiency of filling the cavity are important determinants for specificity. The Kd for imidazole binding increased from 0.7 mM at pH 6 to 3.0 mM at pH 8 and could be fit to a single proton ionization curve with a pKa of 7.4, demonstrating the preferential binding by the imidazolium ion (pKa = 7.3). The binding of a number of substituted imidazoles to the cavity of W191G was verified by X-ray crystallographic analysis. The most clearly defined density was observed for W191G crystals soaked in 1 mM 1,2-dimethylimidazole and was consistent with an oriented occupation in which the unsubstituted nitrogen forms a hydrogen bond or ion pair interaction with Asp-235. Thus, enhanced binding of positively charged molecules may be the result of interactions with this carboxylate. An analogous interaction may stabilize the developing positive charge on the Trp-191 radical of the wild-type enzyme. While the oxidation of imidazoles by the ferryl intermediate of W191G was neither expected nor observed, this study has defined the structural determinants for small molecule binding to an artificially created cavity near a heme center which is capable of generating oxidized species at a potential of over 1 V, and these results will guide future attempts for novel substrate oxidation by CCP.

Two hyperactive mutants of cytochrome c peroxidase (CCP), W51F and W51A, catalyze the enhanced oxidation of a number of substituted anilines. The reaction of CCP compound ES with mesidine is biphasic, while similar reactions using compound II give monophasic kinetics. These data, in addition to the ratio of the Fe4+ = O and free-radical species observed during steady-state turnover, indicate that reduction of the Trp-191 free radical of compound ES is more rapid than the reduction of the Fe4+ = O species. Transient kinetics were examined for the oxidation of eight mono-substituted anilines by CCP, W51F, and W51A. Each of the aniline derivatives were oxidized by the mutants at rates that exceeded that of the wild-type enzyme, and the rate constant for m-chloroaniline was 400-fold faster for W51F than for wild-type CCP. Variations in the rate constants for the different substrates follow a linear free-energy relationship using the Hammet substituent effect parameter sigma +, implicating electron transfer from the aniline ring in the transition state. For aniline oxidation, the free energy of activation is 3 kcal/mol lower for the mutants than for wild-type CCP, and this is due primarily to an increase in the activation entropy. These results indicate that the enhanced kinetics of W51F and W51A result from a generalized increase in enzyme reactivity characterized by an exo-entropic transition state such as dissociation of bound H2O from the Fe4+ = O center.

The buried charge of Asp-235 in cytochrome c peroxidase (CCP) forms an important hydrogen bond to the histidine ligand of the heme iron. The Asp-His-metal interaction, which is similar to the catalytic triad of serine proteases, is found at the active site of many metalloenzymes and is believed to modulate the character of histidine as a metal ligand. We have examined the influence of this interaction in CCP on the function, redox properties, and iron zero-field splitting in the native ferric state and its effect on the Trp-191 free radical site in the oxidized ES complex. Unlike D235A and D235N, the mutation D235E introduces very little perturbation in the X-ray crystal structure of the enzyme active site, with only minor changes in the geometry of the carboxylate-histidine interaction and no observable change at the Trp-191 free radical site. More significant effects are observed in the position of the helix containing residue Glu-235. However, the small change in hydrogen bond geometry is all that is necessary to (1) increase the reduction potential by 70 mV, (2) alter the anisotropy of the Trp-191 free radical EPR, (3) affect the activity and spin-state equilibrium, and (4) reduce the strength of the iron ligand field as measured by the zero-field splitting. The changes in the redox potential with substitution are correlated with the observed zero-field splitting, suggesting that redox control is exerted through the heme ligand by a combination of electrostatic and ligand field effects. The replacement of Asp-235 with Glu appears to result in a significantly weaker hydrogen bond in which the proton resides essentially with His-175. This hydrogen bond is nevertheless strong enough to prevent the reorientation of Trp-191 and the conversion to one of two low-spin states observed for D235A and D235N. The Asp-His-Fe interaction is therefore as important in defining the redox properties and imidazolate character of His-175 as has been proposed, yet its most important role in peroxidase function may be to correctly orient Trp-191 for efficient coupling of the free radical to the heme and to maintain a high-spin 5-coordinate heme center.

An extended accelerated molecular dynamics (AMD) methodology called adaptive AMD is presented. Adaptive AMD (Ad-AMD) is an efficient and robust conformational space sampling algorithm that is particularly-well suited to proteins with highly structured potential energy surfaces exhibiting complex, large-scale collective conformational transitions. Ad-AMD simulations of substrate-free P450cam reveal that this system exists in equilibrium between a fully and partially open conformational state. The mechanism for substrate binding depends on the size of the ligand. Larger ligands enter the P450cam binding pocket, and the resulting substrate-bound system is trapped in an open conformation via a population shift mechanism. Small ligands, which fully enter the binding pocket, cause an induced-fit mechanism, resulting in the formation of an energetically stable closed conformational state. These results are corroborated by recent experimental studies and potentially provide detailed insight into the functional dynamics and conformational behavior of the entire cytochrome-P450 superfamily.

Cytochrome P450 (CYP) 24A1 catalyzes the side-chain oxidation of the hormonal form of vitamin D. Expression of CYP24A1 is up-regulated to attenuate vitamin D signaling associated with calcium homeostasis and cellular growth processes. The development of therapeutics for disorders linked to vitamin D insufficiency would be greatly facilitated by structural knowledge of CYP24A1. Here, we report the crystal structure of rat CYP24A1 at 2.5 A resolution. The structure exhibits an open cleft leading to the active-site heme prosthetic group on the distal surface that is likely to define the path of substrate access into the active site. The entrance to the cleft is flanked by conserved hydrophobic residues on helices A' and G', suggesting a mode of insertion into the inner mitochondrial membrane. A docking model for 1alpha,25-dihydroxyvitamin D(3) binding in the open form of CYP24A1 that clarifies the structural determinants of secosteroid recognition and validates the predictive power of existing homology models of CYP24A1 is proposed. Analysis of CYP24A1's proximal surface identifies the determinants of adrenodoxin recognition as a constellation of conserved residues from helices K, K'', and L that converge with an adjacent lysine-rich loop for binding the redox protein. Overall, the CYP24A1 structure provides the first template for understanding membrane insertion, substrate binding, and redox partner interaction in mitochondrial P450s.

Amino acid replacements of an aromatic residue, Trp-51, which is in contact with the heme of yeast cytochrome c peroxidase have a number of significant effects on the kinetics and coordination state of the enzyme. Six mutants at this site (W51F, W51M, W51T, W51C, W51A, and W51G) were examined. Optical and EPR spectra show that each of these mutations introduces a shift from the 5-coordinate to 6-coordinate form, and slightly increases the asymmetry of the heme ligand field. Conversion from a 6-coordinate high-spin form at pH 5 to a 6-coordinate low-spin form at pH 7 is observed for several of the variants (W51F, W51T, and W51A), while W51G and W51C appear as predominantly low-spin species between pH 5 and 7. Addition of 50% glycerol prevents the facile conversion to the low-spin conformation for W51F, W51T, and W51A, and only W51F can be stabilized in a 5-coordinate configuration by glycerol. For the oxidation of cytochrome c by H2O2, three of the variants (W51F, W51M, and W51T) exhibit values of kcat(app) that are greater than for the wild-type enzyme, while the other mutations give decreased rates of enzyme turnover. Unlike the wild-type enzyme, which functions more efficiently with cytochrome c from yeast than with the horse heart protein, the mutant W51F does not show a preference for substrate from its native organism. The three mutants which exhibit increased values of kcat(app) show a pH optimum at 6.8 compared with that of 5.25 for the wild-type enzyme when measured with horse heart cytochrome c. This shift in pH optimum is not observed with yeast cytochrome c. Construction of single and multiple mutations at Trp-51, Ile-53, and Gly-152 shows that these kinetic properties are not due to natural amino acid variations observed at these sites. Pre-steady-state kinetics show that the bimolecular rate constant for the fast phase of the reaction of the enzyme with H2O2 is only slightly decreased from 3.03 (0.09) X 10(7) to 2.2 (0.1) X 10(7) M-1 s-1 for W51F and to 1.5 (0.1) X 10(7) M-1 s-1 for W51A. The slow phase of the reaction (4.9 s-1) which contributes approximately 30% to the amplitude of the change for the wild-type enzyme is not observed for W51F or W51A.(ABSTRACT TRUNCATED AT 400 WORDS)

A proposed electron transfer pathway in cytochrome c peroxidase was previously excised from the structure by design. The engineered channel mutant was shown to bind peptide surrogates without restoration of cyt c oxidation. Here, we report the 1.6 A crystal structure of (N-benzimidazole-propionic acid)-Gly-Ala-Ala bound within the engineered channel. The peptide retains many features of the native electron transfer pathway: placement of benzimidazole at the position of the Trp-191 radical, hydrogen bonding to Asp235, and positioning of the C-terminus near the point where wild type CcP makes closest contact to cyt c. The inability of this surrogate pathway to restore function supports proposals that electron transfer requires the Trp-191 radical.

Heme-thiolate enzymes, notably cytochromes P450 and nitric oxide synthases, use dioxygen to oxygenatesubstrates. Photoactive metal-diimine molecular wires that are capable of effecting rapid redox state changesat buried active sites have been developed to generate intermediates in the catalytic cycles of these enzymes.Wires that feature a photoactive head group tethered to an active-site ligand bind P450CAM and induciblenitric oxide synthase (iNOS) primarily by hydrophobic interactions. The wire-binding specificity of eachenzyme is critically dependent on the structural flexibility of the protein. P450CAM:wire conjugates canadopt open or partially open conformations, thereby accommodating a wide range of wires, whereas onlylong wires with smaller [Re(CO)3(bpy)Im]+ head groupsare able to bind tightly in the rigid active-site channel of iNOS. Dansyl-terminated molecular wires functionas highly sensitive and isoform specific fluorescent sensors for P450CAM.

A model binding site was used to investigate charge-charge interactions in molecular docking. This simple site, a small (180A(3)) engineered cavity in cyctochrome c peroxidase (CCP), is negatively charged and completely buried from solvent, allowing us to explore the balance between electrostatic energy and ligand desolvation energy in a system where many of the common approximations in docking do not apply. A database with about 5300 molecules was docked into this cavity. Retrospective testing with known ligands and decoys showed that overall the balance between electrostatic interaction and desolvation energy was captured. More interesting were prospective docking scre"ens that looked for novel ligands, especially those that might reveal problems with the docking and energy methods. Based on screens of the 5300 compound database, both high-scoring and low-scoring molecules were acquired and tested for binding. Out of 16 new, high-scoring compounds tested, 15 were observed to bind. All of these were small heterocyclic cations. Binding constants were measured for a few of these, they ranged between 20microM and 60microM. Crystal structures were determined for ten of these ligands in complex with the protein. The observed ligand geometry corresponded closely to that predicted by docking. Several low-scoring alkyl amino cations were also tested and found to bind. The low docking score of these molecules owed to the relatively high charge density of the charged amino group and the corresponding high desolvation penalty. When the complex structures of those ligands were determined, a bound water molecule was observed interacting with the amino group and a backbone carbonyl group of the cavity. This water molecule mitigates the desolvation penalty and improves the interaction energy relative to that of the "naked" site used in the docking screen. Finally, six low-scoring neutral molecules were also tested, with a view to looking for false negative predictions. Whereas most of these did not bind, two did (phenol and 3-fluorocatechol). Crystal structures for these two ligands in complex with the cavity site suggest reasons for their binding. That these neutral molecules do, in fact bind, contradicts previous results in this site and, along with the alkyl amines, provides instructive false negatives that help identify weaknesses in our scoring functions. Several improvements of these are considered.

We report direct electrochemistry of the iNOS heme domain in a DDAB film on the surface of a basal plane graphite electrode. Cyclic voltammetry reveals Fe^(III/II) and Fe^(II/I) couples at −191 and −1049 mV (vs Ag/AgCl). Imidazole and carbon monoxide in solution shift the Fe^(III/II) potential by +20 and +62 mV, while the addition of dioxygen results in large catalytic waves at the onset of Fe^(III) reduction. Voltammetry at higher scan rates (with pH variations) reveals that the Fe^(III/II) cathodic peak can be resolved into two components, which are attributable to Fe^(III/II) couples of five- and six-coordinate hemes. Digital simulation of our experimental data implicates water dissociation from the heme as a gating mechanism for ET in iNOS.

The W191G cavity of cytochrome c peroxidase is useful as a model system for introducing small molecule oxidation in an artificially created cavity. A set of small, cyclic, organic cations was previously shown to bind in the buried, solvent-filled pocket created by the W191G mutation. We docked these ligands and a set of non-binders in the W191G cavity using AutoDock 3.0. For the ligands, we compared docking predictions with experimentally determined binding energies and X-ray crystal structure complexes. For the ligands, predicted binding energies differed from measured values by +/- 0.8 kcal/mol. For most ligands, the docking simulation clearly predicted a single binding mode that matched the crystallographic binding mode within 1.0 A RMSD. For 2 ligands, where the docking procedure yielded an ambiguous result, solutions matching the crystallographic result could be obtained by including an additional crystallographically observed water molecule in the protein model. For the remaining 2 ligands, docking indicated multiple binding modes, consistent with the original electron density, suggesting disordered binding of these ligands. Visual inspection of the atomic affinity grid maps used in docking calculations revealed two patches of high affinity for hydrogen bond donating groups. Multiple solutions are predicted as these two sites compete for polar hydrogens in the ligand during the docking simulation. Ligands could be distinguished, to some extent, from non-binders using a combination of two trends: predicted binding energy and level of clustering. In summary, AutoDock 3.0 appears to be useful in predicting key structural and energetic features of ligand binding in the W191G cavity.