Reduced biopterin as a cofactor in the generation of nitrogen oxides by murine macrophages

Generation of nitric oxide (NO.), an autacoid with vasorelaxant and cytotoxic properties, requires at least three cytosolic components in mouse macrophages besides L-arginine and NADPH. One or more components appear after induction by immunologic stimuli; two or more are present in both activated and non-activated macrophages. The constitutive factors can be separated on a Mr approximately 30,000 cut-off filter into high Mr fraction (HF) and low Mr fraction (LF) (Stuehr, D. J., Kwon, N. S., Gross, S. S., Thiel, B. A., Levi, R., and Nathan, C. F. (1989) Biochem. Biophys. Res. Commun. 161, 420-426). Herein we characterize the major active component in LF. The active component was dialyzable (Mr less than approximately 1,000), water soluble, and cationic at acidic to neutral pH. Fractionation on a C18 column in an acetonitrile/water gradient yielded one broad peak of activity, most of which corresponded to a fluorophore with the excitation/emission spectra of biopterins. Gas chromatography isolated a species in this peak with the mass spectrum of biopterin. Of 14 pteridines tested, only 7,8-dihydrobiopterin (H2biopterin) or 5,6,7,8-tetrahydrobiopterin (H4biopterin) could replace LF in synergizing with HF and the inducible component(s) to generate NO-2 and NO-3, the accumulating oxidation products of NO.. Half-maximal activity required 20-30 nM reduced biopterins. LFs from three cell lines were active in proportion to their content of biopterins; addition of reduced biopterins restored activity to LF from biopterin-deficient cells. Enhancement of NO-2 generation in the presence of H2biopterin but not H4biopterin was abolished by methotrexate and aminopterin, inhibitors of dihydrofolate reductase. These findings implicate a redox cycle in which the generation of NO. is facilitated by catalytic amounts of H4biopterin.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Spectral characterization of brain and macrophage nitric oxide synthases. Cytochrome P-450-like hemeproteins that contain a flavin semiquinone radical.

Nitric oxide (NO) is synthesized in mammals where it acts as a signal molecule for neurotransmission, vasorelaxation, and cytotoxicity. The NO synthases isolated from brain and cytokine-activated macrophages are FAD- and FMN-containing flavoproteins that display considerable sequence homology to NADPH-cytochrome P-450 reductase. However, the nature of their catalytic centers is unknown. We have found that both isoenzymes contain 2 mol of iron-protoporphyrin IX/mol of enzyme homodimer. The optical and EPR spectroscopic properties of the heme groups were found to be remarkably similar to those of high-spin cytochrome P-450. The heme iron in the resting NO synthase is ferric and five-coordinate with a cysteine thiolate as the proximal axial ligand. In addition, the EPR spectra of the resting NO synthases contained a free radical signal attributable to a bound flavin semiquinone that appeared to interact magnetically with the ferric heme iron. NO production was inhibited by carbon monoxide, implying a role for the heme groups in catalysis.     

FAD and GSH participate in macrophage synthesis of nitric oxide.

Following partial purification of macrophage nitric oxide (NO) synthase, enzyme activity requires L-arginine, NADPH, and constitutive cytosolic factors, one of which is tetrahydrobiopterin (BH4) (Kwon, N.S., Nathan, C.F. and Stuehr, D.J. [1989] J. Biol. Chem. 264, 20496). Here we identify FAD and GSH as two additional cofactors needed for full enzyme activity. With all defined cytosolic cofactors in excess, NO synthesis was linear over 3 h and was approximately 50% dependent on exogenous FAD, approximately 50% on glutathione (GSH), 84% on tetrahydrobiopterin (BH4), 95% on NADPH, and 98% on L-arginine. The concentrations of added FAD, GSH, and BH4 required for optimal activity were consistent with their levels in macrophage cytosol. Kinetic studies showed that GSH (or DTT) had little or no effect on the rate of NO generation over the first 20-30 min of the reaction, but prevented a subsequent dropoff in rate. This effect was distinct from thiol participation in BH4 regeneration. In contrast, exogenous FAD doubled the rate of NO synthesis throughout the assay period, consistent with a cofactor role. The role of NADPH was not to regenerate BH4, furnish NADP+, nor form reactive oxygen intermediates. These findings demonstrate NO synthesis by a partially purified enzyme in an otherwise defined system, and suggest that an NADPH-utilizing FAD flavoprotein may participate in the reaction.     

Nitric oxide blocks cellular heme insertion into a broad range of heme proteins

Although the insertion of heme into proteins enables their function in bioenergetics, metabolism, and signaling, the mechanisms and regulation of this process are not fully understood. We developed a means to study cellular heme insertion into apo-protein targets over a 3-h period and then investigated how nitric oxide (NO) released from a chemical donor (NOC-18) might influence heme (protoporphyrin IX) insertion into seven targets that present a range of protein structures, heme ligation states, and functions (three NO synthases, two cytochrome P450's, catalase, and hemoglobin). NO blocked cellular heme insertion into all seven apo-protein targets. The inhibition occurred at relatively low (nM/min) fluxes of NO, was reversible, and did not involve changes in intracellular heme levels, activation of guanylate cyclase, or inhibition of mitochondrial ATP production. These aspects and the range of protein targets suggest that NO can act as a global inhibitor of heme insertion, possibly by inhibiting a common step in the process.     

A Pivotal Role for Tryptophan 447 in Enzymatic Coupling of Human Endothelial Nitric Oxide Synthase (eNOS): EFFECTS ON TETRAHYDROBIOPTERIN-DEPENDENT CATALYSIS AND eNOS DIMERIZATION*

Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by NOS. Bioavailability of BH4 is a critical factor in regulating the balance between NO and superoxide production by endothelial NOS (eNOS coupling). Crystal structures of the mouse inducible NOS oxygenase domain reveal a homologous BH4-binding site located in the dimer interface and a conserved tryptophan residue that engages in hydrogen bonding or aromatic stacking interactions with the BH4 ring. The role of this residue in eNOS coupling remains unexplored. We overexpressed human eNOS W447A and W447F mutants in novel cell lines with tetracycline-regulated expression of human GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, to determine the importance of BH4 and Trp-447 in eNOS uncoupling. NO production was abolished in eNOS-W447A cells and diminished in cells expressing W447F, despite high BH4 levels. eNOS-derived superoxide production was significantly elevated in W447A and W447F versus wild-type eNOS, and this was sufficient to oxidize BH4 to 7,8-dihydrobiopterin. In uncoupled, BH4-deficient cells, the deleterious effects of W447A mutation were greatly exacerbated, resulting in further attenuation of NO and greatly increased superoxide production. eNOS dimerization was attenuated in W447A eNOS cells and further reduced in BH4-deficient cells, as demonstrated using a novel split Renilla luciferase biosensor. Reduction of cellular BH4 levels resulted in a switch from an eNOS dimer to an eNOS monomer. These data reveal a key role for Trp-447 in determining NO versus superoxide production by eNOS, by effects on BH4-dependent catalysis, and by modulating eNOS dimer formation.     

Regulation of inducible nitric oxide synthase by self-generated NO

A ferric heme-nitric oxide (NO) complex can build up in mouse inducible nitric oxide synthase (iNOS) during NO synthesis from L-arginine. We investigated its formation kinetics, effect on catalytic activity, dependence on solution NO concentration, and effect on enzyme oxygen response (apparent KmO2). Heme-NO complex formation was biphasic and was linked kinetically to an inhibition of electron flux and catalysis in iNOS. Experiments that utilized a superoxide generating system to scavenge NO showed that the magnitude of heme-NO complex formation directly depended on the NO concentration achieved in the reaction solution. However, a minor portion of heme-NO complex (20%) still formed during NO synthesis even when solution NO was completely scavenged. Formation of the intrinsic heme-NO complex, and the heme-NO complex related to buildup of solution NO, increased the apparent KmO2 of iNOS by 10- and 4-fold, respectively. Together, the data show heme-NO complex buildup in iNOS is due to both intrinsic NO binding and to equilibrium binding of solution NO, with the latter predominating when NO reaches high nanomolar to low micromolar concentrations. This behavior distinguishes iNOS from the other NOS isoforms and indicates a more complex regulation is possible for its activity and oxygen response in biologic settings.     

Rapid kinetic studies link tetrahydrobiopterin radical formation to heme-dioxy reduction and arginine hydroxylation in inducible nitric-oxide synthase

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.     

Regulation of the properties of the heme-NO complexes in nitric-oxide synthase by hydrogen bonding to the proximal cysteine

Nitric-oxide synthase (NOS) catalyzes the formation of NO and citrulline from l-arginine and oxygen. However, the NO so formed has been found to auto-inhibit the enzymatic activity significantly. We hypothesized that the NO reactivity is in part controlled by hydrogen bonding between the conserved tryptophan residue (position 409 in the neuronal isoform of NOS (nNOS)) and the cysteine residue that forms the proximal bond to the heme. By using resonance Raman spectroscopy and NO as a probe of the heme environment, we show that in the W409F and W409Y mutants of the oxygenase domain of the neuronal enzyme (nNOSox), the Fe-NO bond in the Fe3+NO complex is weaker than in the wild type enzyme, consistent with the loss of a hydrogen bond on the sulfur atom of the proximal cysteine residue. The weaker Fe-NO bond in the W409F and W409Y mutants might result in a faster rate of NO dissociation from the ferric heme in the Trp-409 mutants as compared with the wild type enzyme, which could contribute to the lower accumulation of the inhibitory NO-bound complexes observed during catalysis with the Trp-409 mutants (Adak, S., Crooks, C., Wang, Q., Crane, B. R., Tainer, J. A., Getzoff, E. D., and Stuehr, D. J. (1999) J. Biol. Chem. 274, 26907-26911). The optical and resonance Raman spectra of the Fe2+NO complexes of the Trp-409 mutants differ from those of the wild type enzyme and indicate that a significant population of a five-coordinate Fe2+NO complex is present. These data show that the hydrogen bond provided by the Trp-409 residue is necessary to maintain the thiolate coordination when NO binds to the ferrous heme. Taken together our results indicate that the heme environment on the proximal side of nNOS is critical for the formation of a stable iron-cysteine bond and for the control of the electronic properties of heme-NO complexes.