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In our efforts to develop inhibitors selective for neuronal nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS), we found that nNOS can undergo conformational changes in response to inhibitor binding that does not readily occur in eNOS. One change involves movement of a conserved tyrosine, which hydrogen bonds to one of the heme propionates, but in the presence of an inhibitor, changes conformation, enabling part of the inhibitor to hydrogen bond with the heme propionate. This movement does not occur as readily in eNOS and may account for the reason why these inhibitors bind more tightly to nNOS. A second structural change occurs upon the binding of a second inhibitor molecule to nNOS, displacing the pterin cofactor. Binding of this second site inhibitor requires structural changes at the dimer interface, which also occurs more readily in nNOS than in eNOS. Here, we used a combination of crystallography, mutagenesis, and computational methods to better understand the structural basis for these differences in NOS inhibitor binding. Computational results show that a conserved tyrosine near the primary inhibitor binding site is anchored more tightly in eNOS than in nNOS, allowing for less flexibility of this residue. We also find that the inefficiency of eNOS to bind a second inhibitor molecule is likely due to the tighter dimer interface in eNOS compared with nNOS. This study provides a better understanding of how subtle structural differences in NOS isoforms can result in substantial dynamic differences that can be exploited in the development of isoform-selective inhibitors.

The overproduction of nitric oxide in the brain by neuronal nitric oxide synthase (nNOS) is associated with a number of neurodegenerative diseases. Although inhibiting nNOS is an important therapeutic goal, it is important not to inhibit endothelial NOS (eNOS) because of the critical role played by eNOS in maintaining vascular tone. While it has been possible to develop nNOS selective aminopyridine inhibitors, many of the most potent and selective inhibitors exhibit poor bioavailability properties. Our group and others have turned to more biocompatible thiophene-2-carboximidamide (T2C) inhibitors as potential nNOS selective inhibitors. We have used crystallography and computational methods to better understand how and why two commercially developed T2C inhibitors exhibit selectivity for human nNOS over human eNOS. As with many of the aminopyridine inhibitors, a critical active site Asp residue in nNOS versus Asn in eNOS is largely responsible for controlling selectivity. We also present thermodynamic integration results to better understand the change in p Ka and thus the charge of inhibitors once bound to the active site. In addition, relative free energy calculations underscore the importance of enhanced electrostatic stabilization of inhibitors bound to the nNOS active site compared to eNOS.

The overproduction of nitric oxide in the brain by neuronal nitric oxide synthase (nNOS) is associated with a number of neurodegenerative diseases. Although inhibiting nNOS is an important therapeutic goal, it is important not to inhibit endothelial NOS (eNOS) because of the critical role played by eNOS in maintaining vascular tone. While it has been possible to develop nNOS selective aminopyridine inhibitors, many of the most potent and selective inhibitors exhibit poor bioavailability properties. Our group and others have turned to more biocompatible thiophene-2-carboximidamide (T2C) inhibitors as potential nNOS selective inhibitors. We have used crystallography and computational methods to better understand how and why two commercially developed T2C inhibitors exhibit selectivity for human nNOS over human eNOS. As with many of the aminopyridine inhibitors, a critical active site Asp residue in nNOS versus Asn in eNOS is largely responsible for controlling selectivity. We also present thermodynamic integration results to better understand the change in p Ka and thus the charge of inhibitors once bound to the active site. In addition, relative free energy calculations underscore the importance of enhanced electrostatic stabilization of inhibitors bound to the nNOS active site compared to eNOS.

Development of potent and isoform selective nitric oxide synthase (NOS) inhibitors is challenging because of the structural similarity in the heme active sites. One amino acid difference between NOS isoforms, Asp597 in rat neuronal NOS (nNOS) versus Asn368 in bovine endothelial NOS (eNOS), has been identified as the structural basis for why some dipeptide amide inhibitors bind more tightly to nNOS than to eNOS. We now have found that the same amino acid variation is responsible for substantially different binding modes and affinity for a new class of aminopyridine-based inhibitors.

Leishmania major peroxidase (LmP) is very similar to the well-known yeast cytochrome c peroxidase (CcP). Both enzymes catalyze the peroxidation of cytochrome c. Like CcP, LmP reacts with H2O2 to form Compound I, which consists of a ferryl heme and a Trp radical, Fe(IV)═O;Trp(•+). Cytochrome c (Cytc) reduces the Trp radical to give Compound II, Fe(IV)═O;Trp, which is followed by an intramolecular electron transfer to give Fe(III)-OH;Trp(•+), and in the last step, Cytc reduces the Trp radical. In this study, we have used steady-state and single-turnover kinetics to improve our understanding of the overall mechanism of LmP catalysis. While the activity of CcP greatly increases with ionic strength, the kcat for LmP remains relatively constant at all ionic strengths tested. Therefore, unlike CcP, where dissociation of oxidized Cytc is limiting at low ionic strengths, association/dissociation reactions are not limiting at any ionic strength in LmP. We conclude that in LmP, the intramolecular electron transfer reaction, Fe(IV)═O;Trp to Fe(III)-OH;Trp(•+), is limiting at all ionic strengths. Unlike CcP, LmP depends on key intermolecular ion pairs to form the electron transfer competent complex. Mutating these sites causes the initial rate of association to decrease by 2 orders of magnitude and a substantial decrease in kcat. The drop in kcat is due to a switch in the rate-limiting step of the mutants from intramolecular electron transfer to the rate of association in forming the LmP-LmCytc complex. These studies show that while LmP and CcP form very similar complexes and exhibit similar activities, they substantially differ in how their activity changes as a function of ionic strength. This difference is primarily due to the heavy reliance of LmP on highly specific intermolecular ion pairs, while CcP relies mainly on nonpolar interactions.

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide. N(5)-(1-Iminoethyl)-L-ornithine (L-NIO), an amidine-containing molecule, is a natural product known to be an inactivator of inducible NOS (iNOS). Because of the presence of the amidine methyl group in place of the guanidine amino group of substrate L-arginine, the active site heme peroxy intermediate sometimes cannot be protonated, thereby preventing its conversion to the heme oxo intermediate; instead, a heme oxygenase-type mechanism occurs, leading to conversion of the heme to biliverdin. This might be a new and general inactivation mechanism for heme-containing enzymes. In the studies described here, we attempted to provide support for amidines as substrates and inactivators of iNOS by the design and synthesis of amidine analogues of L-NIO having groups other than the amidine methyl group. No nitric oxide- or enzyme-catalyzed products could be detected by incubation of these amidines with iNOS. Although none of the L-NIO analogues acted as substrates, they all inhibited iNOS; increased inhibitory potency correlated with decreased substituent size. Computer modeling and molecular dynamics simulations were run on 10 and 11 to rationalize why these compounds do not act as substrates. Unlike the methyl amidine (L-NIO), the other alkyl groups block binding of O2 at the heme iron. Compounds 8, 9, and 11 were inactivators; however, no heme was lost, and no biliverdin was formed. No kinetic isotope effect on inactivation was observed with perdeuterated ethyl 8. A small amount of dimer disruption occurred with these inactivators, although the amount would not account for complete enzyme inactivation. The L-NIO analogues inactivate iNOS by a yet unknown mechanism; however, it is different from that of L-NIO, and the inactivation mechanism previously reported for L-NIO appears to be unique to methyl amidines.

Many pyrrolidine-based inhibitors highly selective for neuronal nitric oxide synthase (nNOS) over endothelial NOS (eNOS) exhibit dramatically different binding modes. In some cases, the inhibitor binds in a 180° flipped orientation in nNOS relative to eNOS. From the several crystal structures we have determined, we know that isoform selectivity correlates with the rotamer position of a conserved tyrosine residue that H-bonds with a heme propionate. In nNOS, this Tyr more readily adopts the out-rotamer conformation, while in eNOS, the Tyr tends to remain fixed in the original in-rotamer conformation. In the out-rotamer conformation, inhibitors are able to form better H-bonds with the protein and heme, thus increasing inhibitor potency. A segment of polypeptide that runs along the surface near the conserved Tyr has long been thought to be the reason for the difference in Tyr mobility. Although this segment is usually disordered in both eNOS and nNOS, sequence comparisons and modeling from a few structures show that this segment is structured quite differently in eNOS and nNOS. In this study, we have probed the importance of this surface segment near the Tyr by making a few mutants in the region followed by crystal structure determinations. In addition, because the segment near the conserved Tyr is highly ordered in iNOS, we also determined the structure of an iNOS-inhibitor complex. This new structure provides further insight into the critical role that mobility plays in isoform selectivity.

The nitric oxide synthase (NOS) dimer is stabilized by a Zn(2+) ion coordinated to four symmetry-related Cys residues exactly along the dimer 2-fold axis. Each of the two essential tetrahydrobiopterin (H4B) molecules in the dimer interacts directly with the heme, and each H4B molecule is ~15 Å from the Zn(2+). We have determined the crystal structures of the bovine endothelial NOS dimer oxygenase domain bound to three different pterin analogues, which reveal an intimate structural communication between the H4B and Zn(2+) sites. The binding of one of these compounds, 6-acetyl-2-amino-7,7-dimethyl-7,8-dihydro-4(3H)-pteridinone (1), to the pterin site and Zn(2+) binding are mutually exclusive. Compound 1 both directly and indirectly disrupts hydrogen bonding between key residues in the Zn(2+) binding motif, resulting in destabilization of the dimer and a complete disruption of the Zn(2+) site. Addition of excess Zn(2+) stabilizes the Zn(2+) site at the expense of weakened binding of 1. The unique structural features of 1 that disrupt the dimer interface are extra methyl groups that extend into the dimer interface and force a slight opening of the dimer, thus resulting in disruption of the Zn(2+) site. These results illustrate a very delicate balance of forces and structure at the dimer interface that must be maintained to properly form the Zn(2+), pterin, and substrate binding sites.

A close orthologue to cytochrome P450cam (CYP101A1) that catalyzes the same hydroxylation of camphor to 5-exo-hydroxycamphor is CYP101D1. There are potentially important differences in and around the active site that could contribute to subtle functional differences. Adjacent to the heme iron ligand, Cys357, is Leu358 in P450cam, whereas this residue is Ala in CYP101D1. Leu358 plays a role in binding of the P450cam redox partner, putidaredoxin (Pdx). On the opposite side of the heme, about 15-20 Å away, Asp251 in P450cam plays a critical role in a proton relay network required for O2 activation but forms strong ion pairs with Arg186 and Lys178. In CYP101D1 Gly replaces Lys178. Thus, the local electrostatic environment and ion pairing are substantially different in CYP101D1. These sites have been systematically mutated in P450cam to the corresponding residues in CYP101D1 and the mutants analyzed by crystallography, kinetics, and UV-vis spectroscopy. Individually, the mutants have little effect on activity or structure, but in combination there is a major drop in enzyme activity. This loss in activity is due to the mutants being locked in the low-spin state, which prevents electron transfer from the P450cam redox partner, Pdx. These studies illustrate the strong synergistic effects on well-separated parts of the structure in controlling the equilibrium between the open (low-spin) and closed (high-spin) conformational states.

Research has been conducted on N-alkyl-N'-hydroxyguanidine, characterized as non-amino acid substrates for nitric oxide synthase isoform. The analysis of the crystal structures on nitric oxide synthase heme domain has revealed different binding modes in the substrate binding pocket.

Research has been conducted on cytochrome c peroxidases. The role of the electrostatic destabilization of the cationic radical and the structural and kinetic effects involved in the cytochrome c binding to cytochrome c peroxidase at the surface loop have been investigated and discussed.

Research has been conducted on the nitric oxide which is generated by the isoforms of the nitric oxide synthase under normal conditions. The crystal structure of the catalytic heme domain of the endothelial nitric oxide synthase complexed with its small-molecule inhibitor 3-Br-7-nitroindazole has been investigated and the results are reported.

The structure of nitric oxide synthase complexed with two inhibitors is described. The inhibitors are N(super 5)-(1-iminoethyl)-L-ornithine and N-(3-(aminomethyl)benzyl)acetamide.

One of the most critical phases of O2 activation reaction in bacterial cytochromes P450cam is the chemical process that delivers a proton to the iron-linked dioxygen. The identification of this chemical functionality has been one of the main goals in scientific research. It is believed that a highly conserved carboxylate called aspartate-251 (Asp-251) is a fundamental part of the chemical catalytic process, which the results of experimental studies involving applied kinetic solvent isotope effect methodology to site-directed mutagenesis plus crystallographic analysis strongly suggest.

The threonine residue at position 268 is necessary for the binding and activation of O2 in the metabolism of sodium laurate by cytochrome P450(sub BM-3). Replacement of the residue from Bacillus megaterium reduces the rate of O2 and NADPH utilization. The electron transfer is not linked to substrate hydrolysis, and the production of water and peroxide is higher in the mutant than in the wild-type enzyme. Crystal structure studies indicate that structural changes occur only at the mutation site.

A study was conducted on two X-ray structures of cytochrome P-450(sub CAM) in complex with both enantiomers of an inhibitor made of a chiral center having imidazolylmethyl, dichlorophenyl, hexyl and hydroxyl substituents. The structure of this cytochrome was found to be different from those of its counterparts in that the Tyr96 side chain was oriented upward into the proposed substrate access channel, instead of downward toward the heme. This and other structural changes in the enzyme support existing theories regarding the location of the access channel and suggest possible roles for aromatic residues along this channel for substrate recognition.

The structure of the cytochrome P450cin-substrate complex is reported and compared with the structure of cytochrome P450cam. The cytochrome P450cin uses a flavodoxin-like redox partner to reduce the heme iron rather than more traditional ferrodoxin-like Fe2S2 redox partner used by cytochrome P450cam and many other bacterial p450s.

Heme oxygenation catalyzes the initial steps in heme degradation. The gram negative bacterium pseudomonas aeruginosa contains a heme oxygenase(pa-HO) that primarily oxygenates the Delta-meso heme carbon.

Results demonstrate that X-ray exposure of the crystals of native cytochrome c peroxidase and the reaction intermediate, Compound I, does not reduce iron(IV) but partially reduces the Trp radical. Structural analysis indicate conformational changes mediating the stabilization of Trp191 cationic radical in Compound I.