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We previously reported the existence of a special autoregulation property of neuronal nitric-oxide synthase (NOS) based on NO near-geminate combination and partial trapping of neuronal NOS (nNOS) through a futile regenerating pathway. On this basis, we developed a kinetic simulation model that was proven to predict nNOS catalytic specificities and mutations effects (San- The free radical nitric oxide (NO) 1 is involved in an increasing number of physiologic and pathophysiologic processes (1-6). NO is generated by the NO synthases, which are found in numerous organisms. Animal NO synthases are homodimers whose subunits are comprised of a reductase domain containing FAD, FMN, and NADPH binding sites, and an oxygenase domain containing 6(R)-tetrahydrobiopterin (H 4 B), iron protoporphyrin IX (heme), and a binding site for the L-arginine (Arg) substrate (7,8). NO synthases catalyze two sequential mixedfunction oxidations, the first being Arg hydroxylation to form N -hydroxy-L-arginine (NOHA) as a bound intermediate (9 -11), and the second converting NOHA to citrulline and NO.toliniThree main NO synthases are expressed in mammals and differ in their functions, amino acid sequence, post-translational modification, and cellular location. Two NOS, neuronal NOS (nNOS or NOS-1) and endothelial NOS (eNOS or NOS-3), are constitutively expressed and involved in signal cascades. The third NOS is cytokine-inducible (iNOS or NOS-2) and functions as both a regulator and effector of the immune response. The counterpart to this diversity of location and function is a specific regulation of each isoform. For example, NO synthases differ significantly regarding Ca 2ϩ levels required to bind calmodulin (CaM), which triggers heme reduction and NO synthesis (12, 13). They also have different capacities to be upor down-regulated by serine/threonine phosphorylation (14, 15). A third difference involves regulation via heme⅐NO complex formation. In nNOS, we have established that heme⅐NO complex formation is an intrinsic feature that governs the rate of NO synthesis and shifts the enzyme apparent K m O 2 to a higher value (16,17). The model shown in Scheme 1 below can explain these effects (18). It incorporates the observation that ferric heme binds newly formed NO before it can leave the enzyme (19,20). This causes nNOS to partition between futile and productive cycles during catalysis (see Scheme 1), with only the productive cycle liberating NO (18). Key kinetic parameters for nNOS have been measured, including rates of heme reduction and NO dissociation, k cat , and oxidation of the ferrous heme⅐NO complex (19,21,22). These values are set such that a majority of nNOS exists as the ferrous heme⅐NO complex during steady-state NO synthesis (23,24). Computer simulation of the kinetic model in Scheme 1 reproduces this result and can accurately simulate the pre-steady-state and steady-state behaviors of nNOS mutants that display greater or diminished activity relative to wild-type enzyme (18,25).The available data for iNOS and eNOS suggest r...

Homodimer formation activates all nitric-oxide synthases (NOSs). It involves the interaction between two oxygenase domains (NOSoxy) that each bind heme and (6R)-tetrahydrobiopterin (H4B) and catalyze NO synthesis from L-Arg. Here we compared three NOSoxy isozymes regarding dimer strength, interface composition, and the ability of L-Arg and H4B to stabilize the dimer, promote its formation, and protect it from proteolysis. Urea dissociation studies indicated that the relative dimer strengths were NOSIIIoxy > > NOSIoxy > NOSIIoxy (endothelial NOSoxy (eNOSoxy) > > neuronal NOSOXY (nNOSoxy) > inducible NOSoxy (iNOSoxy)). Dimer strengths of the full-length NOSs had the same rank order as judged by their urea-induced loss of NO synthesis activity. NOSoxy dimers containing L-Arg plus H4B exhibited the greatest resistance to urea-induced dissociation followed by those containing either molecule and then by those containing neither. Analysis of crystallographic structures of eNOSoxy and iNOSoxy dimers showed more intersubunit contacts and buried surface area in the dimer interface of eNOSoxy than iNOSoxy, thus revealing a potential basis for their different stabilities. L-Arg plus H4B promoted dimerization of urea-generated iNOSoxy and nNOSoxy monomers, which otherwise was minimal in their absence, and also protected both dimers against trypsin proteolysis. In these respects, L-Arg alone was more effective than H4B alone for nNOSoxy, whereas for iNOSoxy the converse was true. The eNOSoxy dimer was insensitive to proteolysis under all conditions. Our results indicate that the three NOS isozymes, despite their general structural similarity, differ markedly in their strengths, interfaces, and in how L-Arg and H4B influence their formation and stability. These distinguishing features may provide a basis for selective control and likely help to regulate each NOS in its particular biologic milieu.The free radical nitric oxide (NO) drives important physiologic and patho-physiologic functions in animals and is produced by a family of enzymes termed nitric-oxide synthases (NOSs) 1 (1-4). NOS exists as three main isozymes, inducible NOS (iNOS or Type II), neuronal NOS (nNOS or Type I), and endothelial NOS (eNOS or Type III) as well as their splice variants (5-10). All three NOSs share between 50 and 60% sequence homology (11) and catalyze an NADPH and O 2-dependent oxidation of L-Arg to NO and citrulline, forming N -hydroxy-L-Arg (NOHA) as an intermediate (12, 13). The NOSs exhibit a bidomain structure comprised of an N-terminal oxygenase domain that is linked to a C-terminal reductase domain through a calmodulin (CaM) binding motif (14 -17).Dimerization of NOS proteins is essential for their activity (13, 18 -20). The interaction of two oxygenase domains (NOSoxy) creates an extensive interface between them (21-26). The 6R-tetrahydrobiopterin (H4B) cofactor interacts with residues in both subunits of the dimer and also hydrogen bonds to the active site heme. Dimerization activates NOS in at least three ways: it sequesters heme fro...

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.

Nitric oxide synthases (NOSs) are flavoheme enzymes that contain a ferredoxin:NADP ؉ -reductase (FNR) module for binding NADPH and FAD and are unusual because their electron transfer reactions are controlled by the Ca 2؉ -binding protein calmodulin. A conserved aromatic residue in the FNR module of NOS shields the isoalloxazine ring of FAD and is known to regulate NADPH binding affinity and specificity in related flavoproteins. We mutated Phe-1395 (F1395) in neuronal NOS to Tyr and Ser and tested their effects on nucleotide coenzyme specificity, catalytic activities, and electron transfer in the absence or presence of calmodulin. We found that the aromatic side chain of F1395 controls binding specificity with respect to NADH but does not greatly affect affinity for NADPH. Measures of flavin and heme reduction kinetics, ferricyanide and cytochrome c reduction, and NO synthesis established that the aromatic side chain of F1395 is required to repress electron transfer into and out of the flavins of neuronal NOS in the calmodulin-free state, and is also required for calmodulin to fully relieve this repression. We speculate that the phenyl side chain of F1395 is part of a conformational trigger mechanism that negatively or positively controls NOS electron transfer depending on the presence of calmodulin. Such use of the conserved aromatic residue broadens our understanding of flavoprotein structure and regulation.N itric oxide (NO) is generated by NO synthases (NOSs) and modulates physiology and pathology in mammals (1, 2). The NOS polypeptide consists of an N-terminal oxygenase domain that contains heme, 6R-tetrahydrobiopterin (H 4 B), and an Larginine (Arg) binding site, and a C-terminal reductase domain that contains FMN, FAD, and an NADPH binding site (3-5). An Ϸ20-aa calmodulin (CaM) binding site is located between the reductase and oxygenase domains (3-5). During NO synthesis, electrons from NADPH transfer in a linear sequence to FAD, FMN, and then to the heme, which binds and activates dioxygen (3-5). Although NOS oxygenase domains have unusual structure (6-8), NOS reductase domains are structurally similar to other NADPH-requiring flavoproteins like cytochrome P450 reductase (CYPR), sulfite reductase flavoprotein, methionine synthase reductase, and cytochrome P450BM3 (9-11). These flavoproteins are all comprised of a flavodoxin module that contains FMN and a ferredoxin:NADP ϩ reductase (FNR) module that contains FAD and the NADPH binding site (12).NOS catalysis is controlled by varied mechanisms (3-5, 13). Surprisingly, its electron transfer events are regulated by Ca 2ϩ -dependent CaM binding (14). Although this is unusual for a redox enzyme, it may be representative of a growing class of Ca 2ϩ -activated flavoproteins that include NADPH oxidases (15) and a flavoprotein of Desulfovibrio gigas (16). In NOS, CaM binding relieves a kinetic repression on electron transfer both into and out of its reductase domain (17, 18). We are only beginning to understand the basis for the repression or how it is relieved by ...

A study of the oxidation of a series of guanidines related to L-arginine (L-Arg) and of various alkyl- and arylguanidines, by recombinant NO-synthase II (NOS II), led us to the discovery of the first non-alpha-amino acid guanidine substrate of NOS, acting as an efficient NO precursor. This compound, 3-(trifluoromethyl)propylguanidine, 4, led to a rate of NO formation (k(cat) = 220 +/- 50 min(-1)) only 2 times lower than that of L-Arg. Formation of 1 mol of NO upon NOS II-catalyzed oxidation of 4 occurred with consumption of 2.9 mol of NADPH, which corresponds to a 52% coupling between electron transfer and oxygenation of its guanidine function. Its oxidation by activated mouse macrophages in an L-Arg-free medium resulted in NO(2)(-) formation that was inhibited by classical NOS inhibitors with a rate only 2-3 times lower than that observed with L-Arg itself. These results open the way toward the research of selective, stable guanidine substrates of NOS that could be interesting, new NO donors after in situ oxidation by a given NOS isoform.

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The bc1 complex (bc1) is a redox-driven proton pump of the mitocondrial respiratory chain. Available structures of vertebrate bc1 have insufficient resolution to elucidate the detailed binding of ligand and solvent molecules important for understanding its function. Here we report the 2.5 Å structure of beef bc1 from a new crystal form (unit cell: 144×180×226 Å 3 , P212121). It is now possible to assign water molecules and lipids, and to complete and correct the low resolution models. Details observed at this resolution will be presented and compared with the yeast bc1 structure solved in complex with a Fv fragment. Succinate-ubiquinone oxidoreductase is another membrane protein complex of the respiratory chain, oxidizing succinate into fumarate in the matrix and reducing quinone to quinol in the membrane. The enzyme from chicken heart mitochondria was crystallized in P212121 space group (69×83×291Å 3 ). The data were phased by molecular replacement with a polyalanine model of only the extrinsic part of E. coli fumarate reductase. Positive peaks corresponding to the three iron sulfur clusters, to the FAD, and to the heme of the membrane part were observed in a Fo-Fc map validating the solution. The model is currently being rebuilt in a 3Fo-2Fc map calculated after solvent flattening. It is possible to locate the heme iron and assign some α -helices of the membrane part. An anomalous map at Cu-Kα-wavelength using the current phases shows peaks for the Fe-S clusters and heme. DM43 is an opossum serum glycoprotein inhibitor of snake venom metalloproteinases. It is homologous to human α 1B-glycoprotein, a plasma protein of unknown function and a member of the immunoglobulin supergene family. Size exclusion, dynamic laser light scattering and small angle X-ray scattering (SAXS) data indicate that two monomers of DM43, each composed of three immunoglobulin-like domains, associate to form a homodimer in solution. DM43 inhibits the fibrinogenolytic activities of jararhagin, a PIII snake-venom toxin consisting of metalloproteinase, desintegrin and cysteinerich domains. Evidence suggests that DM43 forms a 1:1 stoichiometric stable complex with jararhagin and that the metalloproteinase domain is essential for such interacion. Homology modeling, based on the crystal structure of a killer cell inhibitory receptor, suggests the existence of an I-type Ig fold for each DM43 domain, a hydrophobic dimerization surface and six exposed loops potentially forming the metalloproteinase binding interface. Jararhagin is shown to have a more compact structure than DM43 with a similar maximum dimension [110(5) Å] but a slightly larger radius of gyration; 34.5(2) Å and 33.7(3) Å respectively. Ab-initio models showed that DM43 is a flattened compact structure whilst Jararhagin is more globular, presenting an internal cavity revealing a structure composed of one large domain and one or two smaller ones. The former probably corresponds to the metalloproteinase domain. These results will be important in the refinement of crystal structures cur...