Intraprotein Electron Transfer in a Two-Domain Construct of Neuronal Nitric Oxide Synthase:  The Output State in Nitric Oxide Formation

Feng, Changjian; Tollin, Gordon; Holliday, Michael A.; Thomas, Clayton; Salerno, John C.; Enemark, John H.; Ghosh, Dipak

doi:10.1021/bi060223n

Cited by 60 publications

(138 citation statements)

References 44 publications

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Section: Nitric Oxide (No)mentioning

confidence: 99%

“…Its interaction with the NOSoxy domain allows the FMN-to-heme electron transfer (10 -17). Current data suggest that conformational equilibria A and B have their own intrinsic set points (K eq ) and individual control (10,12,21), and that the FMN-toheme electron transfer step is fast (12,13,22,23), which implies the conformational kinetic parameters may be rate-limiting for the entire process.In CaM-free NOS, the FMN-shielded conformation of equilibrium A is relatively stable and a crystal structure of the nNOS reductase domain (nNOSr) in this conformation is available (17). CaM binding destabilizes the FMN-shielded conformation and shifts equilibrium A toward the FMN-deshielded form (10,11).…”

mentioning

confidence: 99%

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A Bridging Interaction Allows Calmodulin to Activate NO Synthase through a Bi-modal Mechanism

Tejero¹,

Haque²,

Durra

et al. 2010

Journal of Biological Chemistry

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Calmodulin (CaM) activates the nitric-oxide synthases (NOS) by a mechanism that is not completely understood. A recent crystal structure showed that bound CaM engages in a bridging interaction with the NOS FMN subdomain. We investigated its importance in neuronal NOS (nNOS) by mutating the two residues that primarily create the bridging interaction (Arg 752 in the FMN subdomain and Glu 47 in CaM). Mutations designed to completely destroy the bridging interaction prevented bound CaM from increasing electron flux through the FMN subdomain and diminished the FMN-to-heme electron transfer by 90%, whereas mutations that partly preserve the interaction had intermediate effects. The bridging interaction appeared to control FMN subdomain interactions with both its electron donor (NADPH-FAD subdomain) and electron acceptor (heme domain) partner subdomains in nNOS. We conclude that the Arg 752 -Glu 47 bridging interaction is the main feature that enables CaM to activate nNOS. The mechanism is bi-modal and links a single structural aspect of CaM binding to specific changes in nNOS protein conformational and electron transfer properties that are essential for catalysis. Nitric oxide (NO)4 is an essential signal and effector molecule in biology (1). NO is produced in animals from L-Arg by the NO synthases (NOS, EC 1.14.13.39) (2). Three types of NOS are expressed in mammals: inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) (3-5). The three NOS are structurally homologous and are active as homodimers (6, 7). Each NOS monomer is comprised of two domains: an N-terminal oxygenase domain (NOSoxy) that contains cofactors protoporphyrin IX (heme) and (6R)-5,6,7,8-tetrahydro-L-biopterin (H 4 B) and binds the substrate L-Arg, and a C-terminal reductase domain that contains FAD, FMN, and binds NADPH. The NOS reductase domain is homologous to cytochrome P-450 reductase and related dual-flavin enzymes (5, 8), but also contains up to three regulatory inserts that are unique to the NOS enzymes (3, 5). Significantly, a calmodulin (CaM) binding site is located in the connecting sequence between the NOSoxy and reductase domains (3-5). CaM binding to this site activates NO synthesis by triggering electron transfer to the heme in NOS enzymes (9). The ability of CaM to activate a redox enzyme like NOS is novel and the mechanism is a topic of current interest.Much of the actions of CaM impinge on the NOS FMN subdomain, which is thought to undergo large conformational motions to transfer electrons during catalysis (10 -17). This may be a common feature in the dual-flavin reductase enzyme family (18 -20). Fig. 1 illustrates a model for FMN conformational switching during electron transfer between the FMN and heme within a NOS homodimer. The FMN subdomain must first interact with the NADPH-FAD subdomain (FNR) in a "FMN-shielded" conformation to receive electrons, according to equilibrium A. Once the FMN hydroquinone forms (FMNH 2 ), it must swing away to a "FMN-deshielded" conformation, and then must interact with the NOSoxy do...

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Section: Nitric Oxide (No)mentioning

confidence: 99%

mentioning

confidence: 99%

A Bridging Interaction Allows Calmodulin to Activate NO Synthase through a Bi-modal Mechanism

Tejero¹,

Haque²,

Durra

et al. 2010

Journal of Biological Chemistry

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“…In this model, the FMN domain is suggested to be highly dynamic and flexible due to a connecting hinge that allows it to alternate between its electron-accepting (FAD→FMN) or closed conformation and electron-donating (FMN→heme) or open conformation ( Fig. 1 A and B) (28,(30)(31)(32)(33)(34)(35)(36). In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25,28,32,34,35,37).…”

mentioning

confidence: 99%

“…1 A and B) (28,(30)(31)(32)(33)(34)(35)(36). In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25,28,32,34,35,37). A similar conformational switching mechanism is thought to enable electron transfer through the FMN domain in the related flavoproteins NADPH-cytochrome P450 reductase and methionine synthase reductase (38)(39)(40)(41)(42).…”

mentioning

confidence: 99%

“…1B), and this provides an important layer of regulation (25,27). CaM binding to NOS enzymes increases electron transfer from NADPH through the reductase domain and also triggers electron transfer from the FMN domain to the NOS heme as is required for NO synthesis (31,32). The ability of CaM, or similar signaling proteins, to regulate electron transfer reactions in enzymes is unusual, and the mechanism is a topic of interest and intensive study.…”

mentioning

confidence: 99%

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Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Haque

Stuehr

et al. 2015

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

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Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.A lthough proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein-protein interactions (2-4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5-11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein's structural dynamics link to its functional mechanisms (12-21).A case in point is the nitric oxide synthase (NOS) enzymes (22-24), whose nitric oxide (NO) biosynthesis involves electron transfer reactions that are associated with relatively large-scale movement (tens of Å) of the enzyme domains (Fig. 1A). During catalysis, NADPH-derived electrons first transfer into an FAD domain and an FMN domain in NOS that together comprise the NOS reductase domain (NOSr), and then transfer from the FMN domain to a heme group that is bound in a separate attached "oxygenase" domain, which then enables NO synthesis to begin (22,(25)(26)(27). The electron transfers into and out of the FMN domain are the key steps for catalysis, and they appear to rely on the FMN domain cycling between electron-accepting and electron-donating conformational states (28, 29) (Fig. 1B). In this model, the FMN domain is suggested to be highly...

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Role of an isoform‐specific residue at the calmodulin–heme (NO synthase) interface in the FMN – heme electron transfer

Zheng

Wang

et al. 2018

FEBS Letters

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The interface between calmodulin (CaM) and the NO synthase (NOS) heme domain is the least characterized interprotein interface that the NOS isoforms must traverse through during catalysis. Our previous molecular dynamics simulations predicted a salt bridge between K497 in human inducible NOS (iNOS) heme domain and D118(CaM). Herein, the FMN - heme interdomain electron transfer (IET) rate was found to be notably decreased by charge-reversal mutation, while the IET in the iNOS K497D mutant is significantly restored by the CaM D118K mutation. The results of wild-type protein with added synthetic peptides further demonstrate the critical nature of K497 relative to the rest of the peptide sequence in modulating the IET. These data provide definitive evidence supporting the regulatory role of the isoform-specific K497 residue.

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Intraprotein Electron Transfer in a Two-Domain Construct of Neuronal Nitric Oxide Synthase: The Output State in Nitric Oxide Formation

Cited by 60 publications

References 44 publications

A Bridging Interaction Allows Calmodulin to Activate NO Synthase through a Bi-modal Mechanism

A Bridging Interaction Allows Calmodulin to Activate NO Synthase through a Bi-modal Mechanism

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Role of an isoform‐specific residue at the calmodulin–heme (NO synthase) interface in the FMN – heme electron transfer

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