Charge-pairing interactions control the conformational setpoint and motions of the FMN domain in neuronal nitric oxide synthase

Haque, Mohammad Mahfuzul; Bayachou, Mekki; Fadlalla, Mohammed; Durra, Deborah; Stuehr, Dennis J.

doi:10.1042/bj20121488

Cited by 13 publications

(51 citation statements)

References 50 publications

(131 reference statements)

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“…Expression and Purification of Wild-type and Mutant Proteins-The bovine eNOSr proteins (amino acids 445-1205) were purified by sequential chromatography on a 2Ј,5Ј-ADPSepharose affinity column and CaM-Sepharose resin as reported previously (15,20). Their concentration was determined using an extinction coefficient of 22,900 M Ϫ1 cm Ϫ1 at 457 nm for the fully oxidized form (15,20).…”

Section: Methodsmentioning

confidence: 99%

“…4, the burst kinetics in each panel are due to the rapid reaction of cytochrome c with the subpopulation of eNOSr molecules that are in an open conformation, whose reduced FMN domain reacts with the excess cytochrome c at a rate of about 225 s Ϫ1 (12). The subsequent slower absorbance gain involves the reaction of the remaining eNOSr subpopulation that must open from a closed conformational state in order to react with the cytochrome c. The ratio of these two subpopulations for any given eNOSr protein can be determined from their individual absorbance contributions toward reduction of the first equivalent of cytochrome c and thus are used to determine the eNOSr conformational K eq (15,16,20).…”

Section: Steady-state Cytochrome C Reductase and No Synthesismentioning

confidence: 99%

“…The reactions mixed fully oxidized eNOSr or full-length eNOS proteins with excess NADPH at 10°C under anaerobic conditions in a stopped-flow spectrophotometer, and flavin reduction was followed as an absorbance decrease at 457 nm, whereas heme reduction was followed as build up of the ferrous heme-CO complex at 444 nm (28), respectively. For flavin reduction, all traces fit well to a triple exponential equation as utilized previously (15,29,30), and the fitted rates are listed in Table 2. The initial fast phase k 1 is considered to primarily reflect hydride transfer from NADPH to FAD, followed by slower k 2 and k 3 phases that reflect interflavin electron transfer and NADP ϩ dissociation steps and further reduction by a second NADPH (30,31).…”

Section: Steady-state Cytochrome C Reductase and No Synthesismentioning

confidence: 99%

“…We utilized the stopped-flow traces of cytochrome c reduction in Fig. 4 to estimate the conformational K eq setpoints of the fully reduced wild-type and S1179D eNOSr enzymes in the presence or absence of bound CaM (K eq values are listed in Table 3) (15,16,20). In the reactions of Fig.…”

Section: Steady-state Cytochrome C Reductase and No Synthesismentioning

confidence: 99%

“…1) that links the electron flux through any dual-flavin enzyme to the conformational equilibria and stochastic motions of its FMN domain (18,19). Simulations of this model have proven useful in understanding how conformational aspects regulate ET and catalysis by dual-flavin enzymes (15,16,18,20).Endothelial NO synthase (eNOS) generates NO for cell signaling and functions broadly in health and disease (21-23). Its activity is regulated through several mechanisms, including post-translational modifications that in some cases may control its ET reactions.…”

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confidence: 99%

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Phosphorylation Controls Endothelial Nitric-oxide Synthase by Regulating Its Conformational Dynamics

Haque¹,

Ray²,

Stuehr

2016

Journal of Biological Chemistry

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The activity of endothelial NO synthase (eNOS) is triggered by calmodulin (CaM) binding and is often further regulated by phosphorylation at several positions in the enzyme. Phosphorylation at Ser 1179 occurs in response to diverse physiologic stimuli and increases the NO synthesis and cytochrome c reductase activities of eNOS, thereby enhancing its participation in biological signal cascades. Despite its importance, the mechanism by which Ser 1179 phosphorylation increases eNOS activity is not understood. To address this, we used stopped-flow spectroscopy and computer modeling approaches to determine how the phosphomimetic mutation (S1179D) may impact electron flux through eNOS and the conformational behaviors of its reductase domain, both in the absence and presence of bound CaM. We found that S1179D substitution in CaM-free eNOS had multiple effects; it increased the rate of flavin reduction, altered the conformational equilibrium of the reductase domain, and increased the rate of its conformational transitions. We found these changes were equivalent in degree to those caused by CaM binding to wild-type eNOS, and the S1179D substitution together with CaM binding caused even greater changes in these parameters. The modeling indicated that the changes caused by the S1179D substitution, despite being restricted to the reductase domain, are sufficient to explain the stimulation of both the cytochrome c reductase and NO synthase activities of eNOS. This helps clarify how Ser 1179 phosphorylation regulates eNOS and provides a foundation to compare its regulation by other phosphorylation events.Nitric oxide (NO) synthase enzymes (EC 1.14.13.39) are homodimers with subunits composed of an N-terminal oxygenase domain (NOSoxy) 4 that is linked to a C-terminal reductase domain (NOSr) by an intervening calmodulin (CaM) binding sequence (1-4). Many of the structural and catalytic features of NOSr are shared among a family of eukaryotic dual-flavin enzymes whose members include cytochrome P450 reductase, methionine synthase reductase, and novel reductase-1 (5-7). These enzymes are all composed of an FMN-binding domain that is attached to a FAD/NADPH-binding domain (ferredoxin NADP ϩ -reductase (FNR)) by a flexible hinge of various lengths (8 -10). Their electron transfer (ET) reactions all involve a hydride transfer from NADPH to the FAD cofactor bound within the FNR domain, which then passes electrons sequentially to the FMN domain. Once the FMN domain receives two electrons, its FMN hydroquinone (FMNhq) can transfer an electron to a heme group that is located either within the enzyme itself (i.e. NOS), in a partner hemeprotein acceptor (i.e. cytochrome P450 reductase), or in a non-native hemeprotein acceptor such as cytochrome c (6,(11)(12)(13)(14). Importantly, ET through diflavin enzymes relies on the transient interactions and motions of the FMN domain, which must move between an FNR-bound, conformationally closed "input" state and an unbound, conformationally open "output" state (15-17). We and others have proposed...

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Section: Methodsmentioning

confidence: 99%

Section: Steady-state Cytochrome C Reductase and No Synthesismentioning

confidence: 99%

Section: Steady-state Cytochrome C Reductase and No Synthesismentioning

confidence: 99%

Section: Steady-state Cytochrome C Reductase and No Synthesismentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Phosphorylation Controls Endothelial Nitric-oxide Synthase by Regulating Its Conformational Dynamics

Haque¹,

Ray²,

Stuehr

2016

Journal of Biological Chemistry

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Elucidating nitric oxide synthase domain interactions by molecular dynamics

Hollingsworth

Holden

et al. 2015

Protein Science

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Nitric oxide synthase (NOS) is a multidomain enzyme that catalyzes the production of nitric oxide (NO) by oxidizing l‐Arg to NO and L‐citrulline. NO production requires multiple interdomain electron transfer steps between the flavin mononucleotide (FMN) and heme domain. Specifically, NADPH‐derived electrons are transferred to the heme‐containing oxygenase domain via the flavin adenine dinucleotide (FAD) and FMN containing reductase domains. While crystal structures are available for both the reductase and oxygenase domains of NOS, to date there is no atomic level structural information on domain interactions required for the final FMN‐to‐heme electron transfer step. Here, we evaluate a model of this final electron transfer step for the heme–FMN–calmodulin NOS complex based on the recent biophysical studies using a 105‐ns molecular dynamics trajectory. The resulting equilibrated complex structure is very stable and provides a detailed prediction of interdomain contacts required for stabilizing the NOS output state. The resulting equilibrated complex model agrees well with previous experimental work and provides a detailed working model of the final NOS electron transfer step required for NO biosynthesis.

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A Well-Balanced Preexisting Equilibrium Governs Electron Flux Efficiency of a Multidomain Diflavin Reductase

Frances¹,

Fatemi²,

Pompon³

et al. 2015

Biophysical Journal

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Diflavin reductases are bidomain electron transfer proteins in which structural reorientation is necessary to account for the various intramolecular and intermolecular electron transfer steps. Using small-angle x-ray scattering and nuclear magnetic resonance data, we describe the conformational free-energy landscape of the NADPH-cytochrome P450 reductase (CPR), a typical bidomain redox enzyme composed of two covalently-bound flavin domains, under various experimental conditions. The CPR enzyme exists in a salt- and pH-dependent rapid equilibrium between a previously described rigid, locked state and a newly characterized, highly flexible, unlocked state. We further establish that maximal electron flux through CPR is conditioned by adjustable stability of the locked-state domain interface under resting conditions. This is rationalized by a kinetic scheme coupling rapid conformational sampling and slow chemical reaction rates. Regulated domain interface stability associated with fast stochastic domain contacts during the catalytic cycle thus provides, to our knowledge, a new paradigm for improving our understanding of multidomain enzyme function.

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Charge-pairing interactions control the conformational setpoint and motions of the FMN domain in neuronal nitric oxide synthase

Cited by 13 publications

References 50 publications

Phosphorylation Controls Endothelial Nitric-oxide Synthase by Regulating Its Conformational Dynamics

Phosphorylation Controls Endothelial Nitric-oxide Synthase by Regulating Its Conformational Dynamics

Elucidating nitric oxide synthase domain interactions by molecular dynamics

A Well-Balanced Preexisting Equilibrium Governs Electron Flux Efficiency of a Multidomain Diflavin Reductase

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