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NOSs (NO synthases, EC 1.14.13.39) are haem-thiolate enzymes that catalyse a two-step oxidation of L-arginine to generate NO. The structural and electronic features that regulate their NO synthesis activity are incompletely understood. To investigate how haem electronics govern the catalytic properties of NOS, we utilized a bacterial haem transporter protein to overexpress a mesohaem-containing nNOS (neuronal NOS) and characterized the enzyme using a variety of techniques. Mesohaem-nNOS catalysed NO synthesis and retained a coupled NADPH consumption much like the wild-type enzyme. However, mesohaem-nNOS had a decreased rate of Fe(III) haem reduction and had increased rates for haem-dioxy transformation, Fe(III) haem-NO dissociation and Fe(II) haem-NO reaction with O2. These changes are largely related to the 48 mV decrease in haem midpoint potential that we measured for the bound mesohaem cofactor. Mesohaem nNOS displayed a significantly lower Vmax and KmO2 value for its NO synthesis activity compared with wild-type nNOS. Computer simulation showed that these altered catalytic behaviours of mesohaem-nNOS are consistent with the changes in the kinetic parameters. Taken together, the results of the present study reveal that several key kinetic parameters are sensitive to changes in haem electronics in nNOS, and show how these changes combine to alter its catalytic behaviour.

Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular and cardiac function. Peroxynitrite (ONOO(-)) inactivates eNOS, but questions remain regarding the mechanisms of this process. It has been reported that inactivation is due to oxidation of the eNOS zinc-thiolate cluster, rather than the cofactor tetrahydrobiopterin (BH(4)); however, this remains highly controversial. Therefore, we investigated the mechanisms of ONOO(-)-induced eNOS dysfunction and their dose dependence. Exposure of human eNOS to ONOO(-) resulted in a dose-dependent loss of activity with a marked destabilization of the eNOS dimer. HPLC analysis indicated that both free and eNOS-bound BH(4) were oxidized during exposure to ONOO(-); however, full oxidation of protein-bound biopterin required higher ONOO(-) levels. Additionally, ONOO(-) triggered changes in the UV/visible spectrum and heme content of the enzyme. Preincubation of eNOS with BH(4) decreased dimer destabilization and heme alteration. Addition of BH(4) to the ONOO(-)-destabilized eNOS dimer only partially rescued enzyme function. In contrast to ONOO(-) treatment, incubation with the zinc chelator TPEN with removal of enzyme-bound zinc did not change the eNOS activity or stability of the SDS-resistant eNOS dimer, demonstrating that the dimer stabilization induced by BH(4) does not require zinc occupancy of the zinc-thiolate cluster. While ONOO(-) treatment was observed to induce loss of Zn binding, this cannot account for the loss of enzyme activity. Therefore, ONOO(-)-induced eNOS inactivation is primarily due to oxidation of BH(4) and irreversible destruction of the heme/heme center.

Nitric-oxide synthases (NOS) are heme-thiolate enzymes that N-hydroxylate L-arginine (L-Arg) to make NO. NOS contain a unique Trp residue whose side chain stacks with the heme and hydrogen bonds with the heme thiolate. To understand its importance we substituted His for Trp188 in the inducible NOS oxygenase domain (iNOSoxy) and characterized enzyme spectral, thermodynamic, structural, kinetic, and catalytic properties. The W188H mutation had relatively small effects on l-Arg binding and on enzyme heme-CO and heme-NO absorbance spectra, but increased the heme midpoint potential by 88 mV relative to wild-type iNOSoxy, indicating it decreased heme-thiolate electronegativity. The protein crystal structure showed that the His188 imidazole still stacked with the heme and was positioned to hydrogen bond with the heme thiolate. Analysis of a single turnover L-Arg hydroxylation reaction revealed that a new heme species formed during the reaction. Its build up coincided kinetically with the disappearance of the enzyme heme-dioxy species and with the formation of a tetrahydrobiopterin (H4B) radical in the enzyme, whereas its subsequent disappearance coincided with the rate of l-Arg hydroxylation and formation of ferric enzyme. We conclude: (i) W188H iNOSoxy stabilizes a heme-oxy species that forms upon reduction of the heme-dioxy species by H4B. (ii) The W188H mutation hinders either the processing or reactivity of the heme-oxy species and makes these steps become rate-limiting for l-Arg hydroxylation. Thus, the conserved Trp residue in NOS may facilitate formation and/or reactivity of the ultimate hydroxylating species by tuning heme-thiolate electronegativity.

How 6R-tetrahydrobiopterin (H(4)B) participates in Arg hydroxylation as catalyzed by the nitric oxide synthases (NOSs) is a topic of current interest. Previous work with the oxygenase domain of inducible NOS (iNOSoxy) demonstrated that H(4)B radical formation is kinetically coupled to disappearance of an initial heme-dioxy intermediate and to Arg hydroxylation in a single turnover reaction run at 10 degrees C [Wei, C.-C., Wang, Z.-Q., Wang, Q., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) J. Biol. Chem. 276, 315-319]. Here we used 5-methyl-H(4)B to investigate how pterin structure influences radical formation and associated catalytic steps. In the presence of Arg, the heme-dioxy intermediate in 5-methyl-H(4)B-bound iNOSoxy reacted at a rate of 35 s(-)(1), which is 3-fold faster than with H(4)B. This was coupled to a faster rate of 5-methyl-H(4)B radical formation (40 vs 12.5 s(-)(1)) and to a faster and more productive Arg hydroxylation. The EPR spectrum of the enzyme-bound 5-methyl-H(4)B radical had different hyperfine structure than the bound H(4)B radical and exhibited a 3-fold longer half-life after its formation. A crystal structure of 5-methyl-H(4)B-bound iNOSoxy revealed that there are minimal changes in conformation of the bound pterin or in its interactions with the protein as compared to H(4)B. Together, we conclude the following: (1) The rate of heme-dioxy reduction is linked to pterin radical formation and is sensitive to pterin structure. (2) Faster heme-dioxy reduction increases the efficiency of Arg hydroxylation but still remains rate limiting for the reaction. (3) The 5-methyl group influences heme-dioxy reduction by altering the electronic properties of the pterin rather than changing protein structure or interactions. (4) Faster electron transfer from 5-methyl-H(4)B may be due to increased radical stability afforded by the N-5 methyl group.

In nitric oxide synthase (NOS), (6R)-tetrahydrobiopterin (H(4)B) binds near the heme and can reduce a heme-dioxygen intermediate (Fe(II)O(2)) during Arg hydroxylation [Wei, C.-C., Wang, Z.-Q., Wang, Q., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) J. Biol. Chem. 276, 315-319]. A conserved Trp engages in aromatic stacking with H(4)B, and its mutation inhibits NO synthesis. To examine how this W457 impacts H(4)B redox function, we performed single turnover reactions with the mouse inducible NOS oxygenase domain (iNOSoxy) mutants W457F and W457A. Ferrous mutants containing Arg and H(4)B were mixed with O(2)-containing buffer, and then heme spectral transitions, H(4)B radical formation, and Arg hydroxylation were followed versus time. A heme Fe(II)O(2) intermediate was observed in W457A and W457F and had normal spectral characteristics. However, its disappearance rate (6.5 s(-1) in W457F and 3.0 s(-1) in W457A) was slower than in wild-type (12.5 s(-1)). Rates of H(4)B radical formation (7.1 s(-1) in W457F and 2.7 s(-1) in W457A) matched their rates of Fe(II)O(2) disappearance, but were slower than radical formation in wild-type (13 s(-1)). The extent of H(4)B radical formation in the mutants was similar to wild-type, but their radical decayed 2-4 times faster. These kinetic changes correlated with slower and less extensive Arg hydroxylation by the mutants (wild-type > W457F > W457A). We conclude that W457 ensures a correct tempo of electron transfer from H(4)B to heme Fe(II)O(2), possibly by stabilizing the H(4)B radical. Proper control of these parameters may help maximize Arg hydroxylation and minimize uncoupled O(2) activation at the heme.

To understand how heme and (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)B) participate in nitric-oxide synthesis, we followed ferrous-dioxy heme (Fe(II)O(2)) formation and disappearance, H(4)B radical formation, and Arg hydroxylation during a single catalytic turnover by the inducible nitric-oxide synthase oxygenase domain (iNOSoxy). In all cases, prereduced (ferrous) enzyme was rapidly mixed with an O(2)-containing buffer to start the reaction. A ferrous-dioxy intermediate formed quickly (53 s(-1)) and then decayed with concurrent buildup of ferric iNOSoxy. The buildup of the ferrous-dioxy intermediate preceded both H(4)B radical formation and Arg hydroxylation. However, the rate of ferrous-dioxy decay (12 s(-1)) was equivalent to the rate of H(4)B radical formation (11 s(-1)) and the rate of Arg hydroxylation (9 s(-1)). Practically all bound H(4)B was oxidized to a radical during the reaction and was associated with hydroxylation of 0.6 mol of Arg/mol of heme. In dihydrobiopterin-containing iNOSoxy, ferrous-dioxy decay was much slower and was not associated with Arg hydroxylation. These results establish kinetic and quantitative links among ferrous-dioxy disappearance, H(4)B oxidation, and Arg hydroxylation and suggest a mechanism whereby H(4)B transfers an electron to the ferrous-dioxy intermediate to enable the formation of a heme-based oxidant that rapidly hydroxylates Arg.