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Nitric-oxide synthases (NOS) are catalytically self-sufficient flavo-heme enzymes that generate NO from arginine (Arg) and display a novel utilization of their tetrahydrobiopterin (H4B) cofactor. During Arg hydroxylation, H4B acts as a one-electron donor and is then presumed 1:0 redox cycle (i.e. be reduced back to H4B) within NOS before further catalysis can proceed. Whereas H4B radical formation is well characterized, the subsequent presumed radical reduction has not been demonstrated, and its potential mechanisms are unknown. We investigated radical reduction during a single turnover Arg hydroxylation reaction catalyzed by neuronal NOS to document the process, determine its kinetics, and test for involvement of the NOS flavoprotein domain. We utilized a freeze-quench instrument, the biopterin analog 5-methyl-H~B, and a method that could separately quantify the flavin and iterin radicals that formed in NOS during the reaction. Our results establish that the NOS flavoprotein domain catalyzes reduction of the biopterin radical follow- ing Arg hydroxylation. The reduction is calmodulin-dependent and occurs at a rate that is similar to heme reduction and fast enough to explain H4B redox cycling in NOS. These results, in light of existing NOS crystal structures, suggest a "through- heme" mechanism may operate for H4B radical reduction in NOS. [ABSTRACT FROM AUTHOR]

Unlike animal nitric-oxide synthases (NOSs), the bacterial NOS enzymes have no attached flavoprotein domain to reduce their heme and so must rely on unknown bacterial proteins for electrons. We tested the ability of two Bacillus subtilis flavodoxins (YkuN and YkuP) to support catalysis by purified B. subtilis NOS (bsNOS). When an NADPH-utilizing bacterial flavodoxin reductase (FLDR) was added to reduce YkuP or YkuN, both supported NO synthesis from either L-arginine or N-hydroxyarginine and supported a linear nitrite accumulation over a 30-min reaction period. Rates of nitrite production were directly dependent on the ratio of YkuN or YkuP to bsNOS. However, the V/Km value for YkuN (5.2 x 105) was about 20 times greater than that of YkuP (2.6 X 104), indicating YkuN is more efficient in supporting bsNOS catalysis. YkuN that was either photo-reduced or prereduced by FLDR transferred an electron to the bsNOS ferric heme at rates similar to those measured for heme reduction in the animal NOSs. YkuN supported a similar NO synthesis activity by a different bacterial NOS (Deinococcus radiodurans) but not by any of the three mammalian NOS oxygenase domains nor by an insect NOS oxygenase domain. Our results establish YkuN as a kinetically competent redox partner for bsNOS and suggest that FLDR/flavodoxin proteins could function physiologically to support catalysis by bacterial NOSs. [ABSTRACT FROM AUTHOR]

The nitric-oxide synthases (NOSs) make nitric oxide and citrulline from L-arginine. How the bound cofactor (6R)-tetrahydrobiopterin (H4B) participates in Arg hydroxylation is a topic of interest. We demonstrated previously that H4B radical formation in the inducible NOS oxygenase domain (iNOSoxy) is kinetically coupled to the disappearance of a heme-dioxy intermediate and to Arg hydroxylation. Here we report single turnover studies that determine and compare the kinetics of these transitions in Arg hydroxylation reactions catalyzed by the oxygenase domains of endothelial and neuronal NOSs (eNOSoxy and nNOSoxy). There was a buildup of a heme-dioxy intermediate in eNOSoxy and nNOSoxy followed by a monophasic transition to ferric enzyme during the reaction. The rate of heme-dioxy decay matched the rates of H4B radical formation and Arg hydroxylation in both enzymes. The rates of H4B radical formation differed such that nNOSoxy (18 s-1) > iNOSoxy (11 s-1) > eNOSoxy (6 s-1), whereas the lifetimes of the resulting H4B radical followed an opposite rank order. 5MeH4B supported a three-fold faster radical formation and greater radical stability relative to H4B in both eNOSoxy and nNOSoxy. Our results indicate the following: (i) the three NOSs share a common mechanism, whereby H4B transfers an electron to the heme-dioxy intermediate. This step enables Arg hydroxylation and is rate-limiting for all subsequent steps in the hydroxylation reaction. (ii) A direct correlation exists between pterin radical stability and the speed of its formation in the three NOSs. (iii) Uncoupled NO synthesis often seen for eNOS at low H4B concentrations may be caused by the slow formation and poor stability of its H4B radical. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) release from nitric oxide synthases (NOSs) is largely dependent on the dissociation of an enzyme ferric heme-NO product complex (FeIIINO). Although the NOS-like protein from Bacillus subtilis (bsNOS) generates FeIIINO from the reaction intermediate N-hydroxy-L-arginine (NOHA), its NO dissociation is about 20-fold slower than in mammalian NOSs. Crystal structures suggest that a conserved Val to Ile switch near the heme pocket of bsNOS might determine its kinetic profile. To test this we generated complementary mutations in the mouse inducible NOS oxygenase domain (iNOSoxy, V346I) and in bsNOS (I224V) and characterized the kinetics and extent of their NO synthesis from NOHA and their NO-binding kinetics. The mutations did not greatly alter binding of Arg, (6R)-tetrahydrobiopterin, or alter the electronic properties of the heme or various heme-ligand complexes. Stopped-flow spectroscopy was used to study heme transitions during single turnover NOHA reactions. I224V bsNOS displayed three heme transitions involving four species as typically occurs in wild-type NOS, the beginning ferrous enzyme, a ferrousdioxy (FeIIO2) intermediate, FeIIINO, and an ending ferric enzyme. The rate of each transition was increased relative to wild-type bsNOS, with FeIIINO dissociation being 3.6 times faster. In V346I iNOSoxy we consecutively observed the beginning ferrous, FeIIN2, a mixture of FeIIINO and ferric heme species, and ending ferric enzyme. The rate of each transition was decreased relative to wild-type iNOSoxy, with the FeIIINO dissociation being 3 times slower. An independent measure of NO binding kinetics confirmed that V346I iNOSoxy has slower NO binding and dissociation than wild-type. Citrulline production by both mutants was only slightly lower than wild-type enzymes, indicating good coupling. Our data suggest that a greater shielding of the heme pocket caused by the Val/Ile switch slows down NO synthesis and NO release in NOS, and thus identifies a structural basis for regulating these kinetic variables. [ABSTRACT FROM AUTHOR]

Nitric-oxide synthases are flavoheme enzymes that catalyze two sequential monooxygenase reactions to generate nitric oxide (NO) from L-arginine. We investigated a possible redox role for the enzyme-bound cofactor 6R-tetrahydrobiopterin (H[sub 4]B) in the second reaction of NO synthesis, which is conversion of N-hydroxy-Larginine (NOHA) to NO plus citrulline. We used stopped-flow spectroscopy and rapid-freeze EPR spectroscopy to follow heme and biopterin transformations during single-turnover NOHA oxidation reactions catalyzed by the oxygenase domain of inducible nitric-oxide synthase (iNOSoxy). Significant biopterin radical (> 0.5 per heine) formed during reactions catalyzed by iNOSoxy that contained either H[sub 4]B or 5-methyl-H[sub 4]B. Biopterin radical formation was kinetically linked to conversion of a heme-dioxy intermediate to a berne-NO product complex. The biopterin radical then decayed within a 200300-ms time period just prior to dissociation of NO from a ferric heme-NO product complex. Measures of final biopterin redox status showed that biopterin radical decay occurred via an enzymatic one-electron reduction process that regenerated H[sub 4]B (or 5MeH[sub 4]B). These resuits provide evidence of a dual redox function for biopterin during the NOHA oxidation reaction. The data suggest that H[sub 4]B first provides an electron to a heinedioxy intermediate, and then the H[sub 4]B radical receives an electron from a downstream reaction intermediate to regenerate H[sub 4]B. The first one-electron transition enables formation of the heme-based oxidant that reacts with NOHA, while the second one-electron transition is linked to formation of a ferric heme-NO product complex that can release NO from the enzyme. These redox roles are novel and expand our understanding of biopterin function in biology. [ABSTRACT FROM AUTHOR]

Lenalidomide plays an important role in our chemotherapeutic armamentarium against multiple myeloma, in part by exerting direct anti-proliferative and pro-apoptotic effects. Unfortunately, long-term exposure leads to the development of drug resistance through unknown mechanisms, and we therefore sought to identify pathways that could be responsible for this phenotype. Chronic drug exposure produced myeloma cell lines that were tolerant of the direct effects of lenalidomide, with a degree of resistance of up to 2,500-fold. Gene expression profiling and pathway analysis identified dysregulation of the Wnt/β-catenin pathway as a consistent change across four independent cell isolates, and a pair of primary plasma cell samples. Acute drug treatment also increased β-catenin transcription by 3-fold or more, and both acute and chronic exposure resulted in enhanced accumulation of β-catenin protein by up to 20-fold or more. This produced Wnt/β-catenin pathway activation, as judged by increased activity of a lymphoid enhancer factor/T-cell factor promoter reporter, and enhanced accumulation of the downstream targets cyclin D1 and c-Myc. Components of the β-catenin destruction complex were also impacted by lenalidomide, which suppressed casein kinase 1α expression while augmenting glycogen synthase kinase 3α/β phosphorylation. Stimulation of Wnt/β-catenin signaling with recombinant Wnt-3a, or by overexpression of β-catenin, reduced the anti-proliferative activity of lenalidomide. Conversely, suppression of β-catenin with small hairpin RNAs restored plasma cell sensitivity to lenalidomide. Together, these findings support the hypothesis that lenalidomide mediates activation of Wnt/β-catenin signaling in plasma cells as a mechanism of inducible chemoresistance through effects at the transcriptional and post-translational levels. [ABSTRACT FROM AUTHOR]

Nitric-oxide synthases (NOSs) are calmodulin-dependent flavoheme enzymes that oxidize L-Arg to nitric oxide (No) and L-citrulline. Their catalytic behaviors are complex and are determined by their rates of heme reduction (kr), ferric heme-NO dissociation (kd), and ferrous heme-NO oxidation (kox). We found that point mutation (E762N) of a conserved residue on the enzyme's FMN subdomain caused the NO synthesis activity to double compared with wild type nNOS. However, in the absence of L-Arg, NADPH oxidation rates suggested that electron flux through the heme was slower in E762N nNOS, and this correlated with the mutant having a 60% slower kr During NO synthesis, little heme-NO complex accumulated in the mutant, compared with ∼50-70% of the wild-type nNOS accumulating as this complex. This suggested that the E762N nNOS is hyperactive because it minimizes buildup of an inactive ferrous heme-NO complex during NO synthesis. Indeed, we found that kox was 2 times faster in the E762N mutant than in wild-type nNOS. The mutational effect on kox was independent of calmodulin. Computer simulation and experimental measures both indicated that the slower kr and faster kox of E762N nNOS combine to lower its apparent Km,O2 for NO synthesis by at least 5-fold, which in turn increases its V/Km value and enables it to be hyperactive in steady-state NO synthesis. Our work underscores how sensitive nNOS activity is to changes in the kox and reveals a novel means for the FMN module or protein-protein interactions to alter nNOS activity. [ABSTRACT FROM AUTHOR]

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 importancewesubstitutedHisforTrp188 intheinducibleNOSoxygenase 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 wildtype 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 reductionoftheheme-dioxyspeciesbyH4B.(ii)TheW188Hmutation 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. [ABSTRACT FROM AUTHOR]

Bacterial nitric-oxide (NO) synthases (bNOSs) are smaller than their mammalian counterparts. They lack an essential reductase domain that supplies electrons during NO biosynthesis. This and other structural peculiarities have raised doubts about whether bNOSs were capable of producing NO in vivo. Here we demonstrate that bNOS enzymes from Bacillus subtilis and Bacillus anthracis do indeed produce NO in living cells and accomplish this task by hijacking available cellular redox partners that are not normally committed to NO production. These "promiscuous" bacterial reductases also support NO synthesis by the oxygenase domain of mammalian NOS expressed in Escherichia coli. Our results suggest that bNOS is an early precursor of eukaryotic NOS and that it acquired its dedicated reductase domain later in evolution. This work also suggests that alternatively spliced forms of mammalian NOSs lacking their reductase domains could still be functional in vivo. On a practical side, bNOS-containing probiotic bacteria offer a unique advantage over conventional chemical NO donors in generating continuous, readily controllable physiological levels of NO, suggesting a possibility of utilizing such live NO donors for research and clinical needs. [ABSTRACT FROM AUTHOR]

We studied by ultrafast time-resolved absorption spectroscopy the geminate recombination of NO to the oxygenase domain of the inducible NO synthase, iNOSoxy, and to mutated proteins at position Trp-457. This tryptophan interacts with the tetrahydrobiopterin cofactor BH4, and W457A/F mutations largely reduced the catalytic formation of NO. BH4 decreases the rate of NO rebinding to the ferric iNOSoxy compared with that measured in its absence. The pterin has a larger effect on W457A/F than on the WT protein by increasing NO release from the protein. Therefore, BH4 raises the energy barrier for NO recombination to the mutated proteins in contrast with our observations on eNOS (SlamaSchwok, A., Négrerie, M., Berka, V., Lambry, J.-C., Tsai, A.-L., Vos, M., and Martin, J.-L. (2002) J. Biol. Chem. 277, 7581-7586). Thus, we show a differential effect of BH4 on NO release from eNOS and iNOS. Compared with the position of this residue in the BH4-repleted enzyme, simulations of the NO dissociation dynamics point out at a swing of Trp-457 toward the missing pterin in the absence of BH4. NO geminate-rebinding data show a more efficient NO release from eNOS than from iNOS once NO is formed. Consistently, NO produced by iNOS is regulated by its ferric nitrosyl complex in contrast with eNOS. We show that the small enhancement of the NO geminate recombination rate in W457A/F compared with that in the WT enzyme cannot explain the decrease of NO yield because of the mutation; the major effect of the mutation thus arises from an uncoupled catalysis (Wang, Z. Q., Wei, C. C., Ghosh, S., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) Biochemistry 40, 12819-12825). [ABSTRACT FROM AUTHOR]