Nitric-oxide Synthase Output State

Ghosh, Dipak; Holliday, Michael A.; Clayton, Thomas; Weinberg, J. Brice; Smith, Susan; Salerno, John C.

doi:10.1074/jbc.m509937200

Cited by 52 publications

(69 citation statements)

References 46 publications

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“…4B, the redox potential of the heme was determined by plotting the absorbance at 650 nm versus potential and fitting the data to the one-electron Nernst equation (Equation 2) as explained under "Experimental Procedures." Features of the oxidized FMN are visible around 448 -536 nm, which are further resolved from the heme Soret maxima compared with the situation in the wild-type nNOS bidomain (30). The peaks around 501 nm are mostly from the FMN species.…”

Section: Resultsmentioning

confidence: 82%

“…In the absence of CaM the FMN domain is in close contact with the FAD/NADPH binding domain, a so-called input (closed, shielded) state (30,41,60). This state is locked in by NADPH binding (61) and stabilized by the positions of the C-terminal tail and the autoinhibitory loop in the FMN binding domain seen in the crystal structure of the nNOS reductase domain (54).…”

Section: Discussionmentioning

confidence: 99%

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Exploring the Electron Transfer Properties of Neuronal Nitric-oxide Synthase by Reversal of the FMN Redox Potential

Das

Sibhatu

et al. 2008

Journal of Biological Chemistry

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In nitric-oxide synthase (NOS) the FMN can exist as the fully oxidized (ox), the one-electron reduced semiquinone (sq), or the two-electron fully reduced hydroquinone (hq). In NOS and microsomal cytochrome P450 reductase the sq/hq redox potential is lower than that of the ox/sq couple, and hence it is the hq form of FMN that delivers electrons to the heme. Like NOS, cytochrome P450BM3 has the FAD/FMN reductase fused to the C-terminal end of the heme domain, but in P450BM3 the ox/sq and sq/hq redox couples are reversed, so it is the sq that transfers electrons to the heme. This difference is due to an extra Gly residue found in the FMN binding loop in NOS compared with P450BM3. We have deleted residue Gly-810 from the FMN binding loop in neuronal NOS (nNOS) to give ⌬G810 so that the shorter binding loop mimics that in cytochrome P450BM3. As expected, the ox/sq redox potential now is lower than the sq/hq couple. ⌬G810 exhibits lower NO synthase activity but normal levels of cytochrome c reductase activity. However, unlike the wild-type enzyme, the cytochrome c reductase activity of ⌬G810 is insensitive to calmodulin binding. In addition, calmodulin binding to ⌬G810 does not result in a large increase in FMN fluorescence as in wild-type nNOS. These results indicate that the FMN domain in ⌬G810 is locked in a unique conformation that is no longer sensitive to calmodulin binding and resembles the "on" output state of the calmodulin-bound wild-type nNOS with respect to the cytochrome c reduction activity.Flavin-containing (FMN or FAD) enzymes catalyze a wide range of reactions and can be classified, according to their functions and reactivity with molecular oxygen, into oxidases, monooxygenases, dehydrogenases, oxidoreductases, and electron transferases (1, 2). The versatility of flavoprotein-catalyzed reactions is attributed to the rich chemistry of the flavin isoalloxazine ring system. Free flavin can exist in three different redox states: oxidized (ox), 3 one-electron reduced semiquinoid (sq), and two-electron reduced hydroquinoid (hq) species (3, 4), as shown in Fig. 1. The semiquinone radical can have two forms depending on whether or not the N5 atom is protonated (5). The anionic semiquinone is red, whereas the neutral semiquinone is blue, each with its own distinct UV-visible absorption features (6). The negative charge on the anionic form of the semiquinone or hydroquinone is localized on the N1-C2ϭO group (3). The redox potentials of the ox/sq and sq/hq couples are Ϫ314 mV and Ϫ124 mV, respectively, for free FMN (7), although the FMN redox potentials and the pK a of N5 can vary dramatically from one flavoprotein to another. It is the variations of flavin-protein interactions in different flavoproteins that give rise to the versatility of flavin redox properties tailored to the specific chemical reaction catalyzed by the particular flavoenzyme (2).Before being identified as a heme-containing enzyme (8 -10), nitric-oxide synthase (NOS) was first recognized as a flavoprotein (11, 12). The C-terminal domain of ra...

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Section: Resultsmentioning

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Exploring the Electron Transfer Properties of Neuronal Nitric-oxide Synthase by Reversal of the FMN Redox Potential

Das

Sibhatu

et al. 2008

Journal of Biological Chemistry

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“…Basis for the E762N Mutational Effects on k r and k ox -During nNOS catalysis, the FMN subdomain must oscillate between an interaction with the FNR module to receive electrons and an interaction with the NOSoxy domain to deliver electrons. This involves two separate conformational equilibrium of the FMN subdomain and has been described as a tethered or shuttle mechanism for electron transfer as discussed in detail elsewhere (34,35,37,(42)(43)(44)(45). On the basis of previous studies (20,34,46), an electronegative patch on the FMN subdomain, which includes Glu 762 , is hypothesized to interact with a complementary electropositive patch on the surface of nNOSoxy in order to align the domains and facilitate the FMN-to-heme electron transfer (20).…”

Section: Discussionmentioning

confidence: 99%

Neutralizing a Surface Charge on the FMN Subdomain Increases the Activity of Neuronal Nitric-oxide Synthase by Enhancing the Oxygen Reactivity of the Enzyme Heme-Nitric Oxide Complex

Haque

Fadlalla

Wang

et al. 2009

Journal of Biological Chemistry

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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 (k r ), ferric heme-NO dissociation (k d ), and ferrous heme-NO oxidation (k ox ). 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 k r . During NO synthesis, little heme-NO complex accumulated in the mutant, compared with ϳ50 -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 k ox was 2 times faster in the E762N mutant than in wild-type nNOS. The mutational effect on k ox was independent of calmodulin. Computer simulation and experimental measures both indicated that the slower k r and faster k ox of E762N nNOS combine to lower its apparent K m,O 2 for NO synthesis by at least 5-fold, which in turn increases its V/K m value and enables it to be hyperactive in steady-state NO synthesis. Our work underscores how sensitive nNOS activity is to changes in the k ox and reveals a novel means for the FMN module or protein-protein interactions to alter nNOS activity. Nitric oxide (NO)2 is a biological mediator that is produced in animals by three NO synthase isozymes (NOS, EC 1.14.13.39): inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) (1, 2). The NOS are modular enzymes composed of an N-terminal oxygenase domain and a C-terminal flavoprotein domain, with a calmodulin (CaM)-binding site connecting the two domains (3). During NO synthesis, the flavoprotein domain transfers NADPH-derived electrons through its FAD and FMN cofactors to a heme located in the oxygenase domain. The FMN-to-heme electron transfer enables hemedependent oxygen activation and a stepwise conversion of L-Arg to NO and citrulline (4, 5). Heme reduction also requires that CaM be bound to NOS and is rate-limiting for NO biosynthesis (6 -9).NOS enzymes operate under the constraint of having their newly made NO bind to the ferric heme before it can exit the enzyme (10). How this intrinsic heme-NO binding event impacts NOS catalytic cycling is shown in Fig. 1 and has previously been discussed in detail (10 -13). The L-Arg to NO biosynthetic reaction (Fe III to Fe III NO in Fig. 1) is limited by the rate of ferric heme reduction (k r ), because all biosynthetic steps downstream are faster than k r . However, once the ferric heme-NO complex forms at the end of each catalytic cycle, it can either dissociate to release NO into the medium (at a rate k d as shown in Fig. 1) or become reduced by the flavoprotein domain (at a rate kЈ r in Fig. 1; equal to k r ) to form ...

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“…1A). During catalysis, these hinge elements position the FMN subdomain to receive electrons from the NADPH-FAD module, and then may guide its interactions with the NOS oxygenase domain for electron transfer to the heme (12)(13)(14)(15)(16)(17). In this way, the hinge elements could determine the rate of heme reduction and NO synthesis activity.…”

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confidence: 99%

A connecting hinge represses the activity of endothelial nitric oxide synthase

Haque

Panda

Tejero

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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In mammals, endothelial nitric oxide synthase (eNOS) has the weakest activity, being one-tenth and one-sixth as active as the inducible NOS (iNOS) and the neuronal NOS (nNOS), respectively. The basis for this weak activity is unclear. We hypothesized that a hinge element that connects the FMN module in the reductase domain but is shorter and of unique composition in eNOS may be involved. To test this hypothesis, we generated an eNOS chimera that contained the nNOS hinge and two mutants that either eliminated (P728IeNOS) or incorporated (I958PnNOS) a proline residue unique to the eNOS hinge. Incorporating the nNOS hinge into eNOS increased NO synthesis activity 4-fold, to an activity two-thirds that of nNOS. It also decreased uncoupled NADPH oxidation, increased the apparent K mO2 for NO synthesis, and caused a faster heme reduction. Eliminating the hinge proline had similar, but lesser, effects. Our findings reveal that the hinge is an important regulator and show that differences in its composition restrict the activity of eNOS relative to other NOS enzymes.electron flux ͉ heme reduction ͉ kox N itric oxide (NO) is a widespread signal molecule in biology (1, 2). Three nitric oxide synthases (NOSs) generate NO in mammals [inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS)]. All three are comprised of an Nterminal heme-containing oxygenase domain, an intervening calmodulin (CaM) binding sequence, and a C-terminal reductase domain that contains FMN and FAD (3). The three NOS enzymes have different gene expression patterns, protein interactions, posttranslational modifications, and catalytic behaviors that enable specific roles in biology (4-8). Of note, the NO synthesis activity of eNOS is one-10th that of iNOS and one-sixth that of nNOS. This activity is associated with a slower heme reduction in eNOS (9, 10). Which protein features enable these differences, and why they evolved, are still unclear.Studies of eNOS and nNOS chimeras established that their NO synthesis activities and heme reduction rates are primarily a function of the reductase domain (9, 11). For example, a chimera comprised of an eNOS oxygenase domain fused to an nNOS reductase domain had an NO synthesis activity and heme reduction rate that were similar to those of wild-type nNOS. This result implies that protein structural elements within the reductase domain are largely responsible for the different catalytic behaviors of eNOS and nNOS. While addressing this issue, we became interested in two hinge elements that connect the FMN subdomain of NOS to the rest of the enzyme (Fig. 1A). During catalysis, these hinge elements position the FMN subdomain to receive electrons from the NADPH-FAD module, and then may guide its interactions with the NOS oxygenase domain for electron transfer to the heme (12-17). In this way, the hinge elements could determine the rate of heme reduction and NO synthesis activity.The hinge that connects the FMN subdomain to the rest of the nNOS reductase domain is visible in the reductase crystal structure...

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Nitric-oxide Synthase Output State

Cited by 52 publications

References 46 publications

Exploring the Electron Transfer Properties of Neuronal Nitric-oxide Synthase by Reversal of the FMN Redox Potential

Exploring the Electron Transfer Properties of Neuronal Nitric-oxide Synthase by Reversal of the FMN Redox Potential

Neutralizing a Surface Charge on the FMN Subdomain Increases the Activity of Neuronal Nitric-oxide Synthase by Enhancing the Oxygen Reactivity of the Enzyme Heme-Nitric Oxide Complex

A connecting hinge represses the activity of endothelial nitric oxide synthase

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