Direct Measurement by Laser Flash Photolysis of Intramolecular Electron Transfer in a Two-Domain Construct of Murine Inducible Nitric Oxide Synthase

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

doi:10.1021/ja0578606

Cited by 42 publications

(126 citation statements)

References 24 publications

(48 reference statements)

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“…Thus, the heme reduction rates that we observed in the CT-deleted nNOS provide the best window on how the various mutations at nNOS Arg 752 and CaM Glu 47 affect the formation of FMN-NOSoxy complexes that are productive for electron transfer. Indeed, because the FMN-to-heme electron transfer in a productive complex is very fast (13,22) (Fig. 1), the rates of heme reduction that we measured in the CT-deleted nNOS actually reflect the rates of productive complex formation that occur under each circumstance.…”

Section: Discussionmentioning

confidence: 83%

“…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).…”

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confidence: 99%

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confidence: 99%

“…CaM binding triggers heme reduction in NOS enzymes (9) and so must control the FMN-NOSoxy interaction of equilibrium B. The interaction has been probed by studying electron transfer within partial protein constructs of NOS that contain the linked FMN-CaMNOSoxy domains (12,13,22,23). Although an FMN-NOSoxy interaction was apparent from these studies, the extent of the interaction could not be determined.…”

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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: Discussionmentioning

confidence: 83%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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“…Since this enzyme binds to CaM regardless of cellular Ca 2+ concentrations, this enzyme is classified as Ca 2+ -independent. Electron transfer in the reductase domain of iNOS has previously been determined to be CaMindependent [13]; however, electron transfer from the FMN to the catalytic heme centre in iNOS is CaM dependent [5,14]. The iNOS enzyme can only be purified when coexpressed with CaM and the two proteins remain tightly associated even after the removal of Ca +2 .…”

Section: +mentioning

confidence: 99%

Mapping the Binding and Calmodulin-Dependent Activation of Nitric Oxide Synthase Isozymes

Spratt¹,

Duangkham²,

Taiakina³

et al. 2011

TONOJ

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Calmodulin (CaM) is a ubiquitous cytosolic Ca 2+ -binding protein that can bind and activate numerous intracellular target proteins including the nitric oxide synthase (NOS) enzymes. There are three different isoforms of NOS found in mammals -neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) -with all three isozymes capable of producing the free radical, nitric oxide (•NO). At elevated cellular Ca 2+ concentrations, CaM is able to bind and activate nNOS and eNOS. In contrast, the iNOS isozyme is transcriptionally regulated and binds to CaM in the absence of Ca 2+ . To further investigate the differences in the association of CaM to the Ca 2+ -dependent and Ca 2+ -independent NOS isoforms, a variety of CaM mutants were employed. These included CaM-troponin C chimeras, CaM EF hand pair proteins, CaM mutants incapable of binding to Ca 2+ , methionine oxidized CaM mutants, phosphomimetic CaM mutants and central linker CaM mutants. By characterizing these CaM proteins using a variety of biochemical techniques, we have mapped the binding and the CaM-dependent activation of the NOS enzymes to study their effect on the different electron transfer steps within the NOS enzymes. In addition, fluorescence and Förster resonance energy transfer (FRET) were used to monitor the orientation and conformation of CaM when bound to the NOS peptides and enzymes. Combining these cumulative results, a working model describing the CaM-dependent regulation of iNOS is proposed.

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Intraprotein electron transfer in inducible nitric oxide synthase holoenzyme

et al. 2008

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Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Our previous laser flash photolysis studies provided a direct determination of the kinetics of the FMN-heme IET in a truncated oxyFMN construct of murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along with the calmodulin (CaM) binding site are present [Feng et al. (2006) J. Am. Chem. Soc. 128, 3808-3811]. Here we report the kinetics of the IET in a human iNOS oxyFMN construct, a human iNOS holoenzyme and a murine iNOS holoenzyme, using CO photolysis in comparative studies on partially reduced NOS and a NOS oxygenase construct that lacks the FMN domain. The IET rate constants for the human and murine iNOS holoenzymes are 34 ± 5 s -1 and 35 ± 3 s -1 , respectively, thereby providing a direct measurement of this IET between the catalytically significant redox couples of FMN and heme in the iNOS holoenzyme. These values are approximately an order of magnitude smaller than that in the corresponding iNOS oxyFMN construct, suggesting that in the holoenzyme the rate-limiting step in the IET is the conversion of the shielded electron-accepting (input) state to a new electron-donating (output) state. The fact that there is no rapid IET component in the kinetic traces obtained with the iNOS holoenzyme implies that the enzyme remains mainly in the input state. The IET rate constant value for the iNOS holoenzyme is similar to that obtained for a CaM-bound neuronal NOS (nNOS) holoenzyme, suggesting that CaM activation effectively removes the inhibitory effect of the unique autoregulatory insert in nNOS.

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Direct Measurement by Laser Flash Photolysis of Intramolecular Electron Transfer in a Two-Domain Construct of Murine Inducible Nitric Oxide Synthase

Cited by 42 publications

References 24 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

Mapping the Binding and Calmodulin-Dependent Activation of Nitric Oxide Synthase Isozymes

Intraprotein electron transfer in inducible nitric oxide synthase holoenzyme

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