J. Phys. Chem. B 2009, 113, 3151–3159
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Comparative Computational Analysis of Active and Inactive Cofactors of Nitric Oxide Synthase Do´ra K. Menyha´rd* Department of Inorganic and Analytical Chemistry, Budapest UniVersity of Technology and Economy, Budapest, Szent Gelle´rt te´r 4., H-1111, Hungary ReceiVed: September 18, 2008; ReVised Manuscript ReceiVed: NoVember 28, 2008
Nitric oxide synthases (NOSs) are heme proteins that catalyze the formation of nitric oxide from L-Arg in the presence of oxygen. Of the two electrons required for the first step of the reaction, the second is primarily donated by the tetrahydrobiopterin (H4B) cofactor bound adjacent to the heme, which is eventually reduced back to resting state by the ultimate electron source of the reaction, the flavins of the NOS reductase domain. Density functional theory calculations were carried out to identify those protonation states of different cofactor molecules that best support radicalization of the cofactor and the coupled increase in the electron density of the heme-bound oxygen molecule. Three cofactor molecules were studied, native H4B, an active analogue, 5-methyl-H4B, and the inactive 4-amino-H4B. Findings support the emerging model where H4B and 5-methylH4B are coupled proton/electron sources of NOS catalysis, while 4-amino-H4B is an inhibitor due to its inability to donate the catalytically required proton. Nitric oxide synthases (NOSs) catalyze the formation of nitric oxide from L-Arg in the presence of O2 and nicotinamide adenine dinucleotide phosphate (NADPH). NO produced by inducible NOS (iNOS) in the immune system is a cytotoxic agent, while NO synthesized by endothelial and neuronal NOSs (eNOS, nNOS) in the cardiovascular and neuronal systems serves as a second messenger molecule and activates soluble guanylate cyclase, thereby mediating a number of important physiological processes such as smooth muscle relaxation and neurotransmission.1-4 NOSs consist of two domains: the heme and tetrahydrobiopterin (H4B, shown in Figure 1) binding oxygenase domain, where the catalytic process takes place, and the reductase domain, which binds flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and NADPH cofactors and serves as the electron source of the reaction. The two domains are linked by a flexible, calmodulin (CaM) binding segment. In the cases of eNOS and nNOS, activity is controlled by CaM binding, which results in the two domains moving into proximity, while iNOS has CaM permanently bound to this interdomain region and is under transcriptional control. The sequence identity between the three isoforms reaches 50%, with the sequence and structure of the catalytic oxygenase domain being conserved best.5 To be active, two NOS polypeptides must form a homodimer. The interaction primarily occurs between two oxygenase domains and forms an extensive dimer interface, including the pterin and a Zn2+ ion binding site.5-7 Crystal structures of the oxygenase domain of all three isoforms have been determined.5-9 The formation of NO in the NOS active site is a two-step process, where L-Arg is first converted to Nω-hydroxy-L-Arg (NHA), and in a second step, NHA is further oxidized to NO and L-citrulline, both at the heme site (see Scheme 1.). The steps proceed by similar but not identical mechanisms; the first step is better understood. Once the resting-state ferric heme center is reduced by electron transfer from the reductase domain, it * Tel.: +36-1-4634141. Fax:+36-1-4633408. E-mail: dmenyhard@ mail.bme.hu.
Figure 1. Tetrahydrobiopterin cofactor of NOS.
SCHEME 1: First and Second Steps of the NOS Catalytic Reaction
captures O2, whichsby transfer of a second electronsis turned into FeII-O2-/FeIII-O22-.10 This species accepts two protons, or, as was recently suggested, is a priori doubly protonated,11 and turns into a Compound I type reactant that will attack the substrate.6,12 The second electron is primarily donated by the H4B cofactor, which is eventually reduced back to resting state by the ultimate electron source, the flavins of the reductase domain.13-18 Two different sources of the catalytically required protons of the first reaction step were suggestedsthat of the H4B cofactor10,19-21 and the surrounding bulk solvent11sand several different protonation agents, those of the active site water molecule seen H-bonded to various XO type ligands (NO, CO, and O2) of the heme,11,22,23 the L-Arg substrate itself,6 or an H-bonded network of the two.10 Therefore the details are far from being clarified. It is not yet understood what triggers electron transfer from the cofactor, and where do the implicated protons of the reaction originate from. There are other pteridines that are active cofactors of the NOS catalyzed reactions, while derivatives that are resistant to redox reaction (7,8-dihydropteridines for example) are competitive inhibitors of H4B. The 4-amino analogue of H4B was found to be a potent inhibitor also, in spite of its ability to support dimerization of the enzyme, and the low- to high-spin conversion of the heme just as H4B16,24-26 and its quite similar electro-
10.1021/jp8083056 CCC: $40.75 2009 American Chemical Society Published on Web 02/16/2009
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Figure 2. Proton-transfer pathway connecting the cofactor and the heme site and the most important H-bonding interactions of the cofactor in the crystal structure of iNOS.6
chemical properties,27 and was concluded as such by its inability to serve as a proton donor.21,28,29 In this work we considered three cofactor molecules, native H4B, an active analogue, 5-methyl-H4B, and the inactive 4-amino-H4B. In the full-length enzyme H4B is the most active pterin of the three, followed by 5-methyl-H4B and the inactive 4-amino-H4B. We aimed to identify the species that give rise to the measured radical nature from all chemically plausible protonated and deprotonated forms of each within the NOS active site. Therefore, tautomeric rearrangements and proton transfer between various cofactor proton donors and the heme propionate (a total of 19 different arrangements for the three cofactors) were tested to see if they evoke radicalization of the cofactor in the presence of the heme-bound oxygen molecule, and to see the spin distribution of the radicals produced. The results underline that, in order to ensure the controlled functioning of the enzyme, the finely structured inner region of NOS differentiates between cofactors on the basis not only of their structure and electrochemical properties but also by proton donating capabilities and their ability to successfully participate in the proton-transfer network connecting the cofactor site with the heme centered active region. Methods B3LYP is the most frequently used density functional theory (DFT) method for the study of heme enzymes and has recently been shown especially suited for the description of heme-dioxy complexes;30 however, BP86 was also successfully applied in this field, for example, for describing Compound I models of cytochrome P450 even without the inclusion of noncovalent effects,31 as was necessary when using B3LYP for the same problem.32 Therefore in this study both methods were used in studying H4B and 5-methyl-H4B cofactor containing models, and conclusions were only drawn by trends supported by both, while 4-amino-H4B was only considered using B3LYP. Furthermore, to avoid mistakes arising from the incorrect energetic ordering of spin states, a usual problem of density functional methods, each system was considered in each of the S, T, and Q spin states. Atomic spin densities were calculated by the Mulliken method. All calculations were carried out using the program Jaguar.33 Formulation of the Model Systems. The crystal structure of murine inducible NOS oxygenase domain in complex with H4B6 and 4-amino-H4B34 has been determined; therefore, these structures were used for building our model systems. 5-MeH4B was modeled into the structure of the H4B complex, overlapping H4B atoms. Unified cofactor and heme binding site models were created for the study of radicalization from the respective crystal structures. The heme site contained the entire heme, the oxygen molecule bound to the iron, an SH- group
Figure 3. (A) United model (cofactor and heme site) of the resting state of H4B- and O2-bound NOS (with only polar hydrogens shown). (B-D) Low-energy states obtained in the study of cofactor radicalization (the heme site itself and the indole ring of Trp457 is not shown for clarity). (B) State 3 and state 4 of tetrahydrobiopterin (numbered according to Table 2). (C) State 2 and state 3 of 5-methyltetrahydrobiopterin (numbered according to Table 3). (D) State 3 and state 4 of 4-aminotetrahydrobiopterin (numbered according to Table 4).
standing in place of the proximal Cys residue, anchoring the heme to the protein matrix, the L-Arg substrate, and a water molecule that is usually present in the crystal structures, while the cofactor site consisted of the cofactor molecule, an acetate molecule in place of heme propionate A, and nine waterssfive of which stand in place of various H-bonding partners of the cofactor (those of the guanidino nitrogen of Arg375, and the backbone carbonyls of Ser112, Ile456, Trp457, and Phe470), and the rest are from the crystal structuressand the indole ring of Trp457 which was shown to be essential for enzyme function35 (see Figure 2). Resting-State Calculations. Oxygen Binding to the Heme Site. Using the heme site, the conformation of the heme-bound oxygen, the iron atom, the proximal SH- group, the L-Arg substrate, and an oxygen-close water molecule was optimized using both the UB3LYP and the UBP86 denisty functional method (using the LACV3P** basis set) for the overall spin states of singlet (S), triplet (T), and quintet (Q) in the presence of the fixed atoms of the porphyrin macrocycle, protonated at propionate A to acknowledge the H-bond present in the intact structure toward the pteridine cofactors. Restraining the porphyrin atoms during the calculations can be rationalized by low root mean square (rms) values between NOS porphyrins of so different states. For example, the ligand-free and the NO-bound states of nNOS can be fitted with rms ) 0.10 Å for the porhyrin core non-hydrogen atoms; similarly, the ligand-free and the CObound states with rms ) 0.09 Å (values derived from compari-
Active and Inactive Cofactors of NOS
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TABLE 1: Oxygen Coordinating Geometries at the Heme Sitea B3LYP method
Fe · · · O1 O1-O2 O2 · · · NH1(L-Arg) O2 · · · O(water) Fe · · · S(proximal Cys) Fe-O1-O2 angle a
BP86 method
spin state ) S; rel energy ) 0.0 kJ/mol
spin state ) T; rel energy ) 52.6 kJ/mol
spin state ) Q; rel energy ) 140.5 kJ/mol
spin state ) S; rel energy ) 0.0 kJ/mol
spin state ) T; rel energy ) 0.1 kJ/mol
spin state ) Q; rel energy ) 111.2 kJ/mol
2.06 1.32 2.66 2.91 2.30 125.4
2.08 1.32 2.62 2.89 2.36 125.2
2.11 1.31 2.70 2.94 2.29 125.9
1.90 1.37 2.60 2.84 2.32 126.0
1.91 1.37 2.60 2.83 2.33 129.6
1.95 1.34 2.66 2.89 2.30 129.2
The first five parameters are interatomic distances (Å); the last one is an angle (deg). O1 is the oxygen closer to the heme iron.
TABLE 2: Tetrahydrobiopterin Protonation States, Relative Energies (kJ/mol), and Resultant Spin Densities (Added for the Indicated Atoms) Calculated at Different Overall Spin States Using the B3LYP and BP86 Methodsa,b
a Cofactor spin densities and values belonging to the lowest energy solutions are shown in boldface. b In state 7, energies (marked with an *) are compared just with each other.
son of Protein Data Bank entries 1om4,36 2g6k, and 2g6m22). Binding of the L-Arg substrate to H4B loaded eNOS results in an rms deviation of 0.10 Å for these atoms, while the presence or absence of the H4B cofactor in 0.10 Å (seen when comparing crystal structures 1, 2, and 4 ns 8). The porphyrin structure is preserved even between NOSs of different isoforms; for
example, 0.13 Å rms deviation can be seen between the porphyrins of L-Arg- and H4B-bound eNOS and iNOS (2 nse8 vs 1nod6). Cofactor Binding Site. To consider the resting state of the enzyme, H4B in the neutral form and in the N3 and N5 double-protonated H5B+ form was geometry-optimized within
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TABLE 3: 5-Methyltetrahydrobiopterin Protonation States, Relative Energies (kJ/mol), and Resultant Spin Densities (Added for the Indicated Atoms) Calculated at Different Overall Spin States Using the B3LYP and BP86 Methodsa
a
Cofactor spin densities and values belonging to the lowest energy solutions are shown in boldface.
the cofactor site, adding to the model the guanydino moiety of Arg375 (including CD and CG, the two carbons atoms of the side chain nearest to the guanydino functional group) explicitly. Electron-Transfer between the Heme Bound Oxygen and Various Cofactors. In the calculations pertaining to the radicalization of the cofactor and the coupled changes in the electron density of the heme site, a bias toward radicalization of the cofactors was introduced. The geometry of the cofactor site, belonging to each protonation motif, was optimized (using both density functional methods and the 6-31G** basis set) in the doublet statestherefore these arrangements were preconditioned for hosting a radical. During the optimization the oxygen atoms of the “model” water molecules substituting H-bond partner protein segments, the atoms of the acetate (representing the heme propionate A) and those of the Trp457 ring were kept fixed. This again, can be supported by comparing the crystallographic results. The porphyrin atoms together with the atoms of the indole ring of Trp457 and its carbonyl group, as well as the guaninido group of Arg375, do not show rms deviations exceeding 0.2 Å when the ligation state of the enzyme changes, or upon coordination of its substrate and even between the cofactor-bound and cofactor-free states. The optimized geometries of the cofactor site belonging to each protonation motif were placed into the united model (see Figure 3A), and a single-point energy and spin density calculation was carried out in the S, T, and Q spin states with LACVP** basis (in a recent study even the heptet spin state proved to be of low energy but only in cases when the heme-bound oxygen was doubly protonated11swhich is not the case in any of our model systems). Calculation of the wave function was attempted using several initial guesses. Hypothetical N3 and N5 deprotonated states (except in the case of 5-Me-H4B where only the 3 deprotonation could be carried out) of the cofactors were created where the proton removed from the cofactor was placed on the water molecule above the heme. This, in all cases, resulted in the radicalization of the cofactor, and besides the ligand field theory provided initial guess, these wave functions and those of similar states were also used for generating wave functions of each tautomeric state. Of these, the lowest energy wave function was accepted.
Calculations Testing the Applicability of the Model System. To test the procedure of simply uniting the preoptimized heme and cofactor site models to obtain the spin density distribution of the system, for one selected case, the atoms of the cofactor, the surrounding water molecules, Fe, O2, the L-Arg substrate, the proximal SH- group, and the heme site water molecule were also optimized in the united model containing both sites using UB3LYP/LACVP**. The wave function of the optimized state was also obtained using the LACV3P** basis set for further analysis. To test the effect of restraining porphyrin coordinates, this geometry was further optimized without applying constraints to the atoms of the porphyrin macrocycle. To ensure that the model created produces results in accordance with the rest of the protein matrix, calculations were repeated in the presence of the point charges (according to the AMBER* force field37) of all residues reaching within 12 Å of the cofactor (1117 atoms) for four low-energy states of the united model with the H4B cofactor. Results As will be seen from the results, while using any of the three considered spin states led to essentially identical conclusions, the Q spin state tendentiously proved to be unfavorable, while the S and T states were both of low energy, indicating that the true description of the system is probably a combination of the latter two. We found S statessto different degreessspincontaminated; spin-contamination-free wave functions could only be obtained in the form of high-energy, near closed shell solutions. Although this is a usual problem when using one determinal wave function for describing open shell diradicals,38 it has been shown that unrestricted DFT methods (especially UB3LYP) can give geometries and energetic ordering of such systems that compare well with those using more sophisticated approaches.39-41 We also found that, with the use of the present model system, the UB3LYP method provided more realistic results than UBP86; therefore the third cofactor (4-amino-H4B) was only investigated by this method. Resting-State Calculations. Oxygen Binding to the Heme Site. The oxygen binding geometry of the NOSs has not been so far determined; therefore it had to be calculated. Results are summarized in Table 1. Using either the UB3LYP or the UBP86
Active and Inactive Cofactors of NOS TABLE 4: 4-Aminotetrahydrobiopterin Protonation States, Relative Energies (kJ/mol), and Resultant Spin Densities (Added for the Indicated Atoms) Calculated at Different Overall Spin States Using the B3LYP Methoda
a
Cofactor spin densities and values belonging to the lowest energy solutions are shown in boldface.
method, the singlet state proved to be most stable, although with the UBP86 method the energy difference is marginal. In all structures the oxygen molecule is bound to the heme iron in a bent geometry, maintaining strong H-bonds to both the activesite water molecule and the L-Arg substrate. It was encouraging to note that the results derived with the UB3LYP method are similar to those recently published, where a different model system was used that contained the cofactor too (but missed one of the heme propionates and the active-site water molecule),11 and to our previous results where a slightly smaller model system and basis set were used.42 Cofactor Binding Site. Neutral (H4B), and N3 or N5 doubleprotonated (H5B+) cofactors were geometry-optimized within the cofactor site (including explicitly Arg375). Adding an additional proton to N5 resulted only in slight geometry
J. Phys. Chem. B, Vol. 113, No. 10, 2009 3155 modification as compared to the neutral form, non-hydrogen atoms fitted with 0.2 Å rms. Therefore, based on the structure alone, especially in comparison with the X-ray results, these two states are indistinguishable. On the other hand, when the extra proton of the H5B+ form was added to N3, a proton transfer took place. H+ dissociated from H5B+ and moved to the heme propionate, while H4B shifted 0.4 Å away from the propionate (measured as the distance of their centroids) to avoid the clash of the two hydrogenssthe one passed to the heme propionate and the one remaining on N3sthus resulting in a configuration that can be ruled out on the basis of the crystal structure. Surprisingly, we found that this H4B-H+ state is more favorable than the H5B+ (N5 protonated) form by a formidable 99 kJ/mol. Even though this value is probably an overestimation (since vacuum calculations in such a small, isolated system surely exaggerate the effect of charge-charge repulsion (between Arg375 and the nearby protonated N5)), it is high enough to be significant and demonstrates that, given the chance, H5B+ will readily donate its proton. Thus, our results indicate that binding of tetrahydrobiopterin to NOS is more probable in the neutralsH4Bsform. Electron Transfer between the Heme Bound Oxygen and the Cofactor. For the study of the radicalized state of the model system, the oxygen-bound geometry of the heme site was united with the cofactor site. The cofactor site representing each protonation motif was preoptimized in the doublet state, to introduce a bias toward radicalization through using cofactor geometries consistent with hosting a radical. All results are spincontamination-free solutions, where the cofactor was a full radical (with a sum of atomic spin densities of 1.0). When placing the geometries thus obtained into the united model, one of the following two possibilities took place: (1) within the united model this arrangement did not result in radicalization or (2) the cofactor retained some of its radical character, even within the united model. In this latter case, the spin density distribution within the cofactor was also retained, meaning, that the ratio of the individual atomic spin values and the total spin of the cofactor did not changeswhen compared with the doublet statesonly the total spin of the cofactor. Therefore the spin distribution of even those states that were encumbered with spin contamination (singlet states) reflected those of the more trustworthy solutions. Several protonation motifs were tested; all tautomeric forms corresponding to a neutral resting state (states 1-3 of Table 1 (different tautomers), in case of H4B, for example), which would afford a cation radical after electron withdrawal, and protonation motifs where a proton was removed from the cofactor and placed to either of the heme propionate oxygens, resulting in (in the case of electron transfer) neutral radicals. Tetrahydrobiopterin Cofactor. Results obtained using the united model under vacuum are summarized in Figure 3 and Table 2. In the case of H4B, states 1 and 2, where radicalization of the cofactor would have produced cation radicals, such a shift in the electron density did not take place; neither did the removal of the N3 proton (states 5 and 6) induce radicalization. Partial radicalization of the cofactor took place in states 3 and 4. UB3LYP and UBP86 methods yielded similar results. Both methods indicate that the most stable state of the model created by us is when the proton of the N5 nitrogen of H4B is transferred to the heme propionate, to the oxygen that is directly H-bonded to the proton-transfer pathway to the heme site (state 4, triplet). This arrangement results in partial radicalization of the cofactor too. The fact that only a partial radicalization takes place is not wholly unexpected since, in our model system, the proton
3156 J. Phys. Chem. B, Vol. 113, No. 10, 2009 removed from the cofactor is not actually moved further than either of the carboxylate oxygens of the heme propionate. Therefore, actual proton transfer between the cofactor and the heme-bound ligand or substrate does not take place; neither is the heme site allowed to interact with the extra electron density received from the cofactor. The appearance of 0.48/0.54 spin density at the cofactor is coupled to a -0.43/-0.43 spin density change at the Fe-O2 unit (obtained for the triplet state using the UB3LYP and UBP86 method, respectively)sthe electron density removed from the cofactor transferred to the ferrous-oxy group (with resting-state spin density of 2.00/1.61, using the UB3LYP and UBP86 methods, respectively). The main difference between the results obtained by the two methods is that UBP86 solutions, in all cases, attribute radical character to the heme group, which, however, is in contrast to experimental results. In most cases, however, this only involves those propionate oxygens that are not in connection with the cofactor site (propionate D)sthe negative charge of the carboxylate is neutralized by electron withdrawal from the oxygen atoms. It can also be seen, in neither case did the UBP86 method result in a spin-free state at the cofactor site, but the energetic ordering and the spin value differences between the states, are quite similar to those of UB3LYP. Inclusion of the point-charge field of the amino acid residues reaching within 12 Å of the cofactor did bring a moderation of atomic spin values but did not change the tendencies, resulting in spin values of 0.00, 0.17, 0.38, and 0.00 for the cofactor in states 1, 3, 4, and 6, respectively, supporting corresponding results of the vacuum calculations with a sum of cofactor spin values of 0.00, 0.31, 0.48, and 0.00, respectively. To ensure that the shift in electron density seen between the two sites is not simply the artifact of the applied procedure, the geometry of the most favorable, N5-deprotonated state (state 4) was optimized within the united model system too, using the UB3LYP method. The atoms of the cofactor, the surrounding water molecules, Fe, O2, the L-Arg substrate, the proximal SHgroup, and the heme site water molecule were allowed to rearrange. Optimization did not cause significant change in spin density values; the sum of atomic spin density values of cofactor atoms changed from 0.48 to 0.51/0.45 while those of the Fe-O2 unit from 1.57 to 1.52/1.40 using the LACVP** and the LACV3P** basis set, respectively. This result supported the applicability of our model system and calculation protocol as far as following electron transfer between these two regions by simply the change in atomic spin density values. On the other hand, reoptimization of the heme site in the presence of the cofactor resulted in quite significant geometrical changes. The Fe-O2 distance was lengthened by 0.3 Å, while the distant oxygen atom of the oxygen molecule, H-bonded in the resting state by the L-Arg substrate, was protonated by it. The implicated H, which was originally 1.67 Å from O2 of the oxygen molecule, moved to 1.05 Å of it, the distance of the NH2 nitrogen of L-Arg and the H increased from 1.03 to 1.54 Å. The other NH2 nitrogen of the L-Arg guanydino group, the terminal member of the proton transfer pathway between the cofactor and the heme site, broke from planarity, as if prepared for accepting a proton (see Figure 4). It should be noted, that real proton transfer between the cofactor and the heme site did not take place (it could not, since not all elements of the protontransfer pathway are included even in the united model), the N5 proton of the H4B cofactor was simply placed on the heme propionate, but it seems that, in the case of electron transfer, the L-Arg substrate would be an ideal acceptor of the coupled
Menyha´rd
Figure 4. Optimized geometry belonging to the lowest energy protonation motif of tetrahydrobiopterin with the HOMO of the system also shown (at the isovalue of -0.05). The elongated N · · · H bond of the L-Arg substrate is seen pointing to the heme-bound O2. Carbon atoms of the cofactor are green; nonpolar hydrogen atoms were omitted for clarity.
proton transfer and, as such, could serve as the proton donor of the heme-bound oxygen molecule. To reassure that no artifacts result from restraining porphyrin coordinates, this geometry was further optimized without applying constraints to the atoms of the porphyrin macrocycle. The only notable difference produced by the optimization was the slight flattening of the heme (with a 0.28 Å rms for nonhydrogen heme atoms), resulting in an upward shift of the central iron atom which also caused a 5% shortening of the Fe-O2 bond. Constraint-free optimization of the active site, as could be expected, resulted in an even more complete state of electron transfer between the cofactor and the iron-bound oxygen molecule, with cofactor and Fe-O2 spin of 0.61 and 1.23 (to be compared with the 0.45 and 1.40 values of the constrained optimization). Spin distribution within the cofactor radical did not change, with N5 and N8 participation of 53 and 9% of the total cofactor spin in the constraint-free and 53 and 8% in the constrained optimization, respectively. This shows that, even in the case of cofactor radicalization, when extra electron density is shifted to the Fe-O2 catalytic unit, the model system derived from the resting-state crystal structures and the calculation protocol used by us does not result in great shortcomings while providing a uniform platform for the consideration of various protonation motifs corresponding to different extents of electron transfer from the cofactor site. 5-Methyltetrahydrobiopterin Cofactor. In the case of 5-MeH4B we found that cofactor radicalization is made possible (according to UB3LYP) or is enhanced by (according to UBP86) proton transfer (state 3, singlet; see Table 3 for the vacuum results). Both methods agree that a deprotonated form of the cofactor is energetically favored where the proton removed from the cofactor is transferred to the heme carboxy oxygen directly in contact with the proton-transfer pathway within the enzyme interior. 4-Aminotetrahydrobiopterin Cofactor. At the lowest energy state obtained for 4-amino-H4B (state 3, triplet)swhich, in contrast to that seen in the case of H4B and 5-Me-H4B, is not a deprotonated statesalready modest radicalization of the cofactor and the coupled increase of the electron density of the heme site was found. Radicalization of the cofactor is more characteristic in state 4, that is the second in the stability line (state 4, triplet), where again no proton transfer was carried outstherefore, in both low-energy states a cationic (partial) radical formed. Although transferring a proton from the cofactor to the heme propionate in all cases led to further enhancement of radicalization, all these states proved to be energetically prohibited (see Table 4). Discussion Since experimental results indicate that not only the electron but also the proton donating willingness of the cofactors might
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SCHEME 2: Proposed Reaction Path for the First Step of the NOS Catalytic Reaction
decide if they can support catalysis of NOS, our goal was to see if these two properties can be linked in case of each. The ferrous dioxygen binding geometry and the best arrangement of each tautomeric state to host a radical was calculated in separate heme and cofactor site models, which were then united to see if, by simply placing them next to each other, as seen in the enzyme interior, it will result in an immediate effect. The calculations do not, as they cannot, aim at the mechanism of electron transfer but at finding optimal conditions of it. Pterin radicals produced by single-electron removal were shown to be unstable in aqueous solutions.43 They were, however, extensively analyzed by Westerling and co-workers, both in formic acid and chloroform. From the various 5-methyl pteridine and lumazine derivatives studied, under acidic conditions, mostly N5-centered cationic radicals, while in chloroform, neutralsthough unstablesradicals were formed. Neutral radicals appeared as a result of deprotonation at the N1 or N8 site.44 On the other hand, autooxidation of pteridines, a two-electron process turning H4B into H2B, is believed to involve the N3 proton being transferred to form superoxide.45 Therefore, when studying pterin radicalization within the enzyme interior, several possibilities should be taken into considerationsappearance of the cationic radical as well as N1, N3, N8, or even N5 (if the pterin is unsubstituted at this position) deprotonated neutral radicals could all be expected. In the specific case of H4B-bound NOS, N1 is not protonated and deprotonation at the N8 site is also unlikely, since it is in strong H-bond interaction with the backbone carbonyl of Ile456. It is widely accepted that in the resting state the pterin cofactor should be bound within the enzyme interior in the cationic form (H5B+), presumed to be doubly protonated at N5,46 supported by the finding that, in cofactor-free samples, the positively charged L-Arg itself is captured within the cofactor binding cavity.8 Protonation at this specific spot, however, can be disputed, since the positive charge of the cofactor, instead of benefiting from the favorable electrostatic interaction with the carboxylate group of the heme propionatesas in case of L-Arg binding in the cofactor cavitysis faced with the positive charge of an Arg residue (conserved in all three isoforms), H-bonded via a water molecule to N5, that reaches approximately 4.0 Å of it. On the basis of the comparison of crystal structures of iNOS in complex with active and inactive pterins, Crane et al. concluded in favor of the neutral form also.19 We found that crystallographic results could be consistent with both the N5protonated, cationic, and the neutral form. The N3-protonated form was ruled out, since the proton placed on N3 transferred to the heme propionate during geometry optimization and H4B shifted 0.4 Å away from the propionate, creating a conformation no longer reconcilable with the crystallographic results. Sørlie et al. proposed that the cofactor itself could be the initial source of the proton required for catalysis and described a proton-transfer pathway starting from N3 of the cofactor, H-bonded to the heme propionate, on to the terminal amino
group of the L-Arg substrate, H-bonded to the carboxylate oxygen of Glu371, which in turn reaches two of the guanidino nitrogens (NE, NH2) of the L-Arg substrate.21 However, in crystal structures of active pterins bound to various isomers of NOS, this route can be extended to the N5 nitrogen also, via the 4-keto group of the cofactor and two structural water molecules (see Figure 2). The water molecule above the N5 nitrogen is fixed by a highly conserved Arg residue (Arg375 in murine iNOS), the same residue that H-bonds the 4-keto group of the H4B cofactor. Mutating this Arg to an apolar residue, even though the same heme environment was able to form in the presence of the L-Arg substrate, resulted in the reduction of the NO generating ability of eNOS to 9%,47 in the case of iNOS, to 25% of that of the wild-type.48 Since six further H-bonds and a stacking interaction with Trp457 secures the H4B cofactor within the protein matrix, it is unlikely that its destabilization alone (loosing H-bond to the 4-keto group) would be able to account for such loss of enzymatic activity; rather these findings might underline the significance of the water molecule which provides a proton-transfer route from N5 to the heme-bound substrate. Therefore both N3 and N5 are plausible candidates as deprotonation sites; radicalization coupled to proton transfer would result in a cation or a neutral radical, depending on the resting-state protonation motif. The fact that autooxidation (proceeding through loss of the N3 hydrogen) is suppressed within the protein matrix17 indicates that N3 deprotonation of H4B is less probable in this environment. According to our results H4B radicalization can be most successfully evoked by N5 deprotonation. Neither placing the cationic or the neutral cofactor (in a geometry optimized to host a radical) in the same system with the oxygen-loaded heme site nor transferring the N3 proton to either carboxylate oxygens of the heme propionate resulted in electron density being shifted from the cofactor to the heme-bound ligand. It is important to note that the calculations were attempted using preradicalized guess wave functions too, where the SCF procedure was started from a solution describing the cofactor as a radical, but this approach also lead to spin-free final solutions for the cofactor atoms in each case. Radical character appears when the proton of N5 is merely transferred from nitrogen to the 4-keto group of the neutral cofactor (state 3 of Table 2); however, it is enhanced and is energetically more favorable when the proton is placed onto the heme propionateson the oxygen directly linked to the proton-transfer pathway leading to the heme site (state 4 of Table 2). The former results in an N5-centered radical cation (with a charge of +0.7), while the latter is an N5-centered neutral radical, which means that the majority of the spin density is carriedsin both casessby N5, in agreement with the experimental results. Satisfactory simulation of the EPR signal of the pterin radical appearing in NOS catalysis has been achieved by a number of different approaches, using three, five, or seven nuclei to model the obtained results. A minimal requirement was found to be
3158 J. Phys. Chem. B, Vol. 113, No. 10, 2009 one nitrogen and two proton nuclei (N5, N5-HR, and C6-Hβ)21,49 to match the data; Wei et al. found the best fit using two nitrogen nuclei and three protons (N5, N8, N5-HR, N8-Hβ, and C6-Hβ),50 while Schmidt et al. included two further protons in their simulation set (those of C7-Hβ1 and C7-Hβ2). For good reproduction of the experimental results, the authors found that the N5 spin value had to be kept between 0.4 and 0.5 while that of N8 around 0.1.51 The EPR results agree in the radical being a protonated, N5-centered cation radical. However, the solution-state pKa of the H4B•+ radical cation was measured to be 5.2, which indicates it would rapidly deprotonate at physiological pH.52 Our results show that after formation of a cation radical (with N5 and N8 atomic spin densities of 61 and 6% of the total spin density of the cofactor in the vacuum calculation and 58 and 6% when surrounded with the point charges of the protein), it will relax by passing the proton to the proton-transfer pathway connecting the cofactor site with the heme-bound oxygen molecule, resulting in a neutral radical (with N5 and N8 atomic spin densities of 53 and 8% of the total, both in the vacuum calculation and when surrounded by the point charges of the protein bulk). Although N5 is deprotonated in both models, this might be reconciled with the observed EPR signal considering that N5 is in a rather tight H-bond with the water molecule above it in both structures, and especially in state 4, where the N5 · · · Hwater distance is only 1.8 Å (see Figure 3). Electron transfer assisted by proton transfer along such a long path might also seem as a too strict requirement for a realistic process, but H4B electron transfer is the second the slowest step in the biosynthesis of NO (the slowest is electron transfer from the reducing domain)12swhich might allow for a complex mechanism. 5-Methyl-H4B binds weaker to the oxygenase domain of NOS and is a less potent activator of the catalytic process in the fulllength enzyme than H4B,53 while in kinetic studies using just the heme-containing “oxy” domain of iNOS and eNOS, it transferred the electron to the heme-bound oxygen faster than H4B.50,54 Either way, it does support NO formation and is radicalized during the reaction with oxygen. The radical signal emerging is similar in shape and line width to those of H4B; characteristic differences appear in its the hyperfine structure.50,53 Since only three protons were required to fit the EPR data, the 5-methyl-H4B pterin radical was concluded to be neutral.50 Our findings agree; the radical appears as a result of the N3 proton being transferred to the heme propionate, along the H-bond present between these two, and becomes more characteristic and energetically more favorable when it is moved even further to the distant carboxylate oxygen (states 2 and 3 of Table 3). The spin distribution of the radical is similar to that seen in the N5-deprotonated H4B, the main difference being that while in N5-deprotonated H3B•, the approximately 60% of the spin content is carried by N5 alone, in 3-deprotonated (5-methylH3B)•, it is split between N5 and the neighboring carbon atom (23 and 46%), while N8 carries a spin of approximately 10% (7%) of all, just as in the case of H3B•. Solution pKa of 4-amino-H4B is 6.755 as compared with the 5.6 value for that of H4B.56 In the NOS environment, tautomeric equilibrium of 4-amino-H4B is believed to be shifted by the proximity of the heme propionate (2.66 Å) toward the form, where N3 carries a proton, resulting in an imine on C4.18 This is reflected in the position of the Arg375 group also, which H-bonds this nitrogen with both of its terminal guanidino nitrogens, while the implicated water molecule, connecting the N5 nitrogen of the cofactor to the proton-transfer network, shifts from above the N5 nitrogen closer to the 4-imino group, so
Menyha´rd that H-bonding to N5 issin the crystal structureslost (however, it is probably easily restored within the fully solvated physiological state). In our results, this imine tautomer of 4-aminoH4B (state 3 of Table 4) indeed proved to be energetically most favorable and partially radicalized without prior rearrangement of its protonation motifsin other words, simply the presence of the oxy-heme induced an electron shift. Another tautomeric form, where the 4-amino group is retained and N3 remains protonated at the expense of the N5 nitrogen (state 4 of Table 4)salso a low-energy statesresults in further enhancement of radicalization. On the other hand, all forms of proton transfer between the cofactor and the heme propionate studied by us proved to be energetically quite unfavorable, which, accepting that an active cofactor should serve as a proton donor also, is in accordance with the observation that, in the presence of 4-amino-H4B, the catalytic reaction does not proceed beyond the reduction of the heme-bound oxygen.21,28 The overall spin motif is similar in this case again; in the lowest energy state, state 3, N5 and the adjacent carbon carry 68% of the spin density of the cofactor (33% at N5, 35% at C4a), while the spin density of N8 is 9% of the total. In state 4, spin density is shifted toward the N5 nitrogen, N5 carrying 54% of the spin density of the cofactor alone, while C4a only 5% and N8 7%. The resemblance among spin distribution of the three different pteridine cofactor radicals is even more interesting in light of the fact that radical formation in this case is coupled to yet another protonation motif. On the basis of the results, it can be proposed that while, in case of H4B and 5-methyl-H4B, the active cofactors of NOS electron-transfer to the heme site are coupled to proton transfer, in the case of the inactive 4-amino- H4B it is not. H4B will not oxidize when the NOS heme is unligated or the NO product is bound to it; a unique feature of the ferrous-oxy/ ferric-superoxy complex triggers its radicalization. According to our results this might well be its proton affinity. In a recent DFT study, Cho and co-workers demonstrated11 that the protonation state of the active site is directly linked to the electron-donating affinity of the H4B cofactor and suggested that radicalization of the cofactor will take place only after the spontaneous double protonation of the heme-bound oxygen molecule. This model, however, does not explain why 4-aminoH4B, quite able and successful in electron donation,21,28 cannot support the catalytic reaction. Our results, on the other hand, indicate that a complementary effect is also true, namely, that the protonation state of the cofactor affects the spin density at the heme site, which, taken together with the results of Cho and co-workers suggests that a synergism exists between the two. This notion is supported by optimizing the heme-bound ligand, substrate and the N5-deprotonated cofactor within the same system, which led to proton transfer between the L-Arg substrate and the oxygen molecule, showing in our model system, too, that protonation of the heme-bound oxygen stabilizes the cofactor radical. In the enzyme interior, where the proton removed from the cofactor can transfer to the substrate via the proton-transfer pathway connecting the twos creating a highly potent proton donor from L-Argsthe protonation of the heme-bound oxygen by the substrate is even more plausible (see Scheme 2). Conclusion In case of the three different cofactors studied, radicalization was easiest to evoke by characteristically different protonation motifs. NOS on one hand seems architectured to withstand these variations, however providing a keen control mechanism also, where not only members of the protein matrix but also the
Active and Inactive Cofactors of NOS structural water molecules play a crucial role in selecting the appropriate cofactor to support the catalytic reaction. Our results indicate that, in the case of H4B and 5-methyl-H4B, radicalization is assisted by proton transfer, but, in the case of 4-aminoH4B, we found that while electron donation from the cofactor to the heme site is possible, proton transfer is hindered. As the transient acceptor of the proton originating from the cofactor, the L-Arg substrate was shown to be a plausible protonation agent of the heme-bound oxygen molecule. Findings support the emerging model where H4B and 5-methyl-H4B are coupled proton/electron sources of NOS catalysis,21,28,29 while 4-aminoH4B is an inhibitor due to its inability to donate the catalytically required proton. Acknowledgment. D.K.M. is a Bolyai research fellow of the Hungarian Academy of Sciences, which is gratefully aknowleged. References and Notes (1) Palmer, R. M.; Ferrige, A. G.; Moncada, S. Nature 1987, 327, 524. (2) Moncada, S.; Palmer, R. M.; Higgs, E. A. Pharmacol. ReV. 1991, 43, 109. (3) Nathan, C. J. Clin. InVest. 1997, 100, 2417. (4) Li, H.; Poulos, T. L. J. Inorg. Biochem. 2005, 99, 293. (5) Fischmann, T. O.; Hruza, A.; Niu, X. D.; Fossetta, J. D.; Lunn, C. A.; Dolphin, E.; Prongay, A. J.; Reichert, P.; Lundell, D. J.; Narula, S. K.; Weber, P. C. Nat. Struct. Biol. 1999, 6, 233. (6) Crane, B. R.; Arvai, A. S.; Ghosh, D. K.; Wu, C.; Getzoff, E. D.; Stuehr, D. J.; Tainer, J. A. Science 1998, 279, 2121. (7) Li, H.; Raman, C. S.; Glaser, C. B.; Blasko, E.; Zoung, T. A.; Parkinson, J. F.; Whitlow, M.; Poulos, T. L. J. Biol. Chem. 1999, 274, 21276. (8) Raman, C. S.; Li, H.; Marta´sek, P.; Kra´l, V.; Masters, B. S. S.; Poulos, T. L. Cell 1998, 95, 939. (9) Li, H.; Shimizu, H.; Flinspach, M.; Jamal, J.; Yang, W.; Xian, M.; Cai, T.; Wen, E. Z.; Qiang Jia, Q.; Wang, P. G.; Poulos, T. L. Biochemistry 2002, 41, 13868. (10) Davydov, R.; Ledbetter-Rogers, A.; Marta´sek, P.; Larukhin, M.; Sono, M.; Dawson, J. H.; Masters, B. S. S.; Hoffman, B. M. Biochemistry 2002, 41, 10375. (11) Cho, K. B.; Derat, E.; Shaik, S. J. Am. Chem. Soc. 2007, 129, 3182. (12) Stuehr, D. J.; Santolini, J.; Wang, Z. Q.; Wei, C. C.; Adak, S. J. Biol. Chem. 2004, 279, 36167. (13) Hurshman, A. R.; Krebs, C.; Edmondson, D. E.; Huynh, B. H.; Marletta, M. A. Biochemistry 1999, 38, 15689. (14) Bec, N.; Gorren, A. F. C.; Mayer, B.; Schmidt, P. P.; Andersson, K. K.; Lange, R. J. Inorg. Biochem. 2000, 81, 207. (15) Wei, C. C.; Wang, Z. Q.; Wang, Q.; Meade, A. L.; Hemann, C.; Hille, R.; Stuehr, D. J. J. Biol. Chem. 2001, 276, 315. (16) Wei, C. C.; Wang, Z. Q.; Hemann, C.; Hille, R.; Stuehr, D. J. J. Biol. Chem. 2003, 278, 46668. (17) Berka, V.; Yeh, H. C.; Gao, D.; Kiran, F.; Tsai, A. L. Biochemistry 2004, 43, 13137. (18) Ost, T. W.; Daff, S. J. Biol. Chem. 2005, 280, 965. (19) Crane, B. R.; Arvai, A. S.; Ghosh, S.; Getzoff, E. D.; Stuehr, D., J.; Tainer, J. A. Biochemistry 2000, 39, 4608. (20) Gorren, A. C.; Mayer, B. Curr. Drug. Metab. 2002, 3, 133. (21) Sørlie, M.; Gorren, A. C.; Marchal, S.; Shimizu, T.; Lange, R.; Andersson, K. K.; Mayer, B. J. Biol. Chem. 2003, 278, 48602.
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