A conserved flavin-shielding residue regulates NO synthase electron transfer and nicotinamide coenzyme specificity

Adak, Subrata; Sharma, Manisha; Meade, Abigail L.; Stuehr, Dennis J.

doi:10.1073/pnas.192283399

Cited by 46 publications

(73 citation statements)

References 38 publications

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“…Altering the C terminus may loosen a tightly tuned electron transfer system, so that either deletion of the unique tails or phosphorylation increases flavin reduction and consequently NO activity in a CaM-independent manner. Further supporting this argument, mutation of the FNR-family conserved C-terminal aromatic residue in nNOS similarly increases electron transfer rates in the absence of CaM (27).…”

Section: And References Therein)mentioning

confidence: 64%

Nitric-oxide Synthase (NOS) Reductase Domain Models Suggest a New Control Element in Endothelial NOS That Attenuates Calmodulin-dependent Activity

Knudsen

Nishida

Mooney

et al. 2003

Journal of Biological Chemistry

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Inducible (iNOS) and constitutive (eNOS, nNOS) nitric-oxide synthases differ in their Ca2؉ -calmodulin (CaM) dependence. iNOS binds CaM irreversibly but eNOS and nNOS, which bind CaM reversibly, have inserts in their reductase domains that regulate electron transfer. These include the 43-45-amino acid autoinhibitory element (AI) that attenuates electron transfer in the absence of CaM, and the C-terminal 20 -40-amino acid tail that attenuates electron transfer in a CaMindependent manner. We constructed models of the reductase domains of the three NOS isoforms to predict the structural basis for CaM-dependent regulation. We have identified and characterized a loop (CD2A) within the NOS connecting domain that is highly conserved by isoform and that, like the AI element, is within direct interaction distance of the CaM binding region. The eNOS CD2A loop (eCD2A) has the sequence 834 KGSPG-GPPPG 843 , and is truncated to 809 ESGSY 813 (iCD2A) in iNOS. The eCD2A contributes to the Ca 2؉ dependence of CaM-bound activity to a level similar to that of the AI element. The eCD2A plays an autoinhibitory role in the control of NO, and CaM-dependent and -independent reductase activity, but this autoinhibitory function is masked by the dominant AI element. Finally, the iCD2A is involved in determining the salt dependence of NO activity at a post-flavin reduction level. Electrostatic interactions between the CD2A loop and the CaM-binding region, and CaM itself, provide a structural means for the CD2A to mediate CaM regulation of intra-subunit electron transfer within the active NOS complex.Nitric-oxide synthase (NOS), 1 a modular protein, consists of an N-terminal heme domain that catalyzes P450-like oxidations and a C-terminal two-flavin domain homologous to that of cytochrome P450 reductase (CPR) (1, 2). The NOS oxygenase and reductase domains are linked by a calmodulin (CaM) binding helix. The NOS oxygenase domain is completely different from a P450, however, in that it has no sequence identity with that family of enzymes and has an ␣,␤-fold structure distinct from the highly ␣-helical structure of the P450 enzymes (3-6). Furthermore, the two-stage mechanism for the oxidation of L-arginine to L-citrulline and nitric oxide (NO) by NOS is more complex than that of a P450 enzyme, as it requires tetrahydrobiopterin (H 4 B) as a transient electron donor (7). In contrast, the NOS reductase domain exhibits considerable sequence identity with CPR, and the electrons, as in P450/CPR, flow from NADPH to the FAD, then to the FMN, and finally, upon substrate binding, to the heme (2, 8).In the cytochrome P450 system, the principal control on inter-protein electron transfer is the requirement for association of the P450 with CPR. The NOS complex also requires interactions between independently transcribed proteins because it is a homodimer in which the reductase domain of one subunit provides electrons to the oxygenase domain of the other (9, 10). However, inter-domain electron transfer in NOS also requires the binding of CaM, and the Ca 2ϩ...

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Section: And References Therein)mentioning

confidence: 64%

Nitric-oxide Synthase (NOS) Reductase Domain Models Suggest a New Control Element in Endothelial NOS That Attenuates Calmodulin-dependent Activity

Knudsen

Nishida

Mooney

et al. 2003

Journal of Biological Chemistry

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“…Mutations of the homologous residue (F1395) in nNOS (62) to the nonaromatic residue serine, but not to the aromatic residue tyrosine, affected cytochrome c, DCIP, and ferricyanide reduction and flavin reduction kinetics, confirming that the aromaticity of the residue in this position is essential. In addition, and unlike wild-type nNOS, the Phe-1395-Ser mutant increased its activity with NADH, which eliminates the 2Ј-phosphate as a factor in binding interactions.…”

Section: Discussionmentioning

confidence: 89%

Recruitment of governing elements for electron transfer in the nitric oxide synthase family

Jáchymová

Martásek

Panda

et al. 2005

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

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At least three building blocks are responsible for the molecular basis of the modulation of electron transfer in nitric oxide synthase (NOS) isoforms: the calmodulin-binding sequence, the C-terminal extension, and the autoregulatory loop in the reductase domain. We have attempted to impart the control conferred by the C termini of NOS to cytochrome P450 oxidoreductase (CYPOR), which contains none of these regulatory elements. The effect of these C termini on the properties of CYPOR sheds light on the possible evolutionary origin of NOS and addresses the recruitment of new peptides on the development of new functions for CYPOR. The C termini of NOSs modulate flavoprotein-mediated electron transfer to various electron acceptors. The reduction of the artificial electron acceptors cytochrome c, 2,6-dichlorophenolindophenol, and ferricyanide was inhibited by the addition of any of these C termini to CYPOR, whereas the reduction of molecular O 2 was increased. This suggests a shift in the rate-limiting step, indicating that the NOS C termini interrupt electron flux between flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and͞or the electron acceptors. The modulation of CYPOR by the addition of the NOS C termini is also supported by flavin reoxidation and fluorescence-quenching studies and antibody recognition of the C-terminal extension. These experiments support the origin of the NOS enzymes from modules consisting of a heme domain and CYPOR or ferredoxin-NADP ؉ reductase-and flavodoxin-like subdomains that constitute CYPOR, followed by further recruitment of smaller modulating elements into the flavin-binding domains.flavoprotein ͉ NADPH-cytochrome P450 reductase ͉ protein evolution

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“…This is an important distinction because point mutation of a nearby conserved residue has broader effects. Specifically, mutation of the aromatic residue Phe-1395 not only altered the ability of nNOS to discriminate between NADPH and NADH but also relieved the repression of electron transfer and catalysis that is normally observed in the CaM-free state (59). Thus, although Asp-1393 and Phe-1395 lie just upstream in sequence from the CaM-responsive C-terminal regulatory element in nNOS (Fig.…”

Section: Fig 5 Kinetics Of Heme Reduction In D1393v Nnos At 10°cmentioning

confidence: 92%

A Conserved Aspartate (Asp-1393) Regulates NADPH Reduction of Neuronal Nitric-oxide Synthase

Panda¹,

Adak²,

Konas

et al. 2004

Journal of Biological Chemistry

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Nitric-oxide synthases (NOSs) are flavo-heme enzymes whose electron transfer reactions are controlled by calmodulin (CaM). The NOS flavoprotein domain includes a ferredoxin-NADP ؉ reductase (FNR)-like module that contains NADPH-and FADbinding sites. FNR-like modules in related flavoproteins have three conserved residues that regulate electron transfer between bound NAD(P)H and FAD. To investigate the function of one of these residues in neuronal NOS (nNOS), we generated and characterized mutants that had Val, Glu, or Asn substituted for the conserved Asp-1393. All three mutants exhibited normal composition, spectral properties, and binding of cofactors, substrates, and CaM. All had slower NADPH-dependent cytochrome c and ferricyanide reductase activities, which were associated with proportionally slower rates of NADPH-dependent flavin reduction in the CaM-free and CaM-bound states. Rates of NO synthesis were also proportionally slower in the mutants and were associated with slower rates of CaMdependent ferric heme reduction. However, a D1393V mutant whose flavins had been prereduced with NADPH had a normal rate of heme reduction. This indicated that the kinetic defect was restricted to flavin reduction step(s) in the mutants and suggested that this limited their catalytic activities. Together, our results show the following. 1) The presence and positioning of the Asp-1393 carboxylate side chain are critical to enable NADPHdependent reduction of the nNOS flavoprotein. 2) Control of flavin reduction is important because it ensures that the rate of heme reduction is sufficiently fast to enable NO synthesis by nNOS. Nitric oxide (NO)1 is a widespread signal and effector molecule in biology (1-3). Animals generate NO by virtue of the nitric-oxide synthases (NOSs), which catalyze a stepwise, NADPH-dependent oxidation of L-Arg to NO and L-citrulline, with N -hydroxy-L-arginine (NOHA) being formed as an intermediate. Each NOS is composed of an N-terminal oxygenase domain that binds iron protoporphyrin IX (heme), (6R)-tetrahydrobiopterin (H 4 B), and substrate, L-Arg, and a C-terminal flavoprotein domain that binds FAD, FMN, and NADPH. A calmodulin (CaM)-binding sequence connects the oxygenase and flavoprotein domains. During catalysis, electrons from NADPH transfer into the FAD and FMN groups of the NOS flavoprotein domain, and then transfer one at a time from the FMN hydroquinone (FMNH 2 ) to the ferric heme (4 -8) (Scheme 1).Heme reduction enables the enzyme to bind O 2 and initiate an H 4 B-dependent oxygen activation that is required for catalysis (9, 10). In all NOSs studied so far, the rates of NADPHdependent flavin reduction are fast relative to the rates of electron transfer from FMNH 2 to the ferric heme in the CaMbound enzymes (11)(12)(13)(14). This makes heme reduction rate-limiting for NO synthesis and implies that the NOS flavins predominantly exist in their reduced forms during steady-state NO synthesis.NOSs are part of a small enzyme family whose members have covalently linked flavoprotein and heme domain...

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A conserved flavin-shielding residue regulates NO synthase electron transfer and nicotinamide coenzyme specificity

Cited by 46 publications

References 38 publications

Nitric-oxide Synthase (NOS) Reductase Domain Models Suggest a New Control Element in Endothelial NOS That Attenuates Calmodulin-dependent Activity

Nitric-oxide Synthase (NOS) Reductase Domain Models Suggest a New Control Element in Endothelial NOS That Attenuates Calmodulin-dependent Activity

Recruitment of governing elements for electron transfer in the nitric oxide synthase family

A Conserved Aspartate (Asp-1393) Regulates NADPH Reduction of Neuronal Nitric-oxide Synthase

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