Mutation in the Flavin Mononucleotide Domain Modulates Magnetic Circular Dichroism Spectra of the iNOS Ferric Cyano Complex in a Substrate-Specific Manner

Sempombe, Joseph; Galinato, Mary Grace I.; Elmore, Bradley O.; Fan, Weihong; Guillemette, J. Guy; Lehnert, Nicolai; Kirk, Martin L.; Feng, Changjian

doi:10.1021/ic200952c

Cited by 12 publications

(25 citation statements)

References 30 publications

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“…Additionally, the MCD spectroscopic perturbation observed with l -Arg is absent in the CN − complexes incubated with NOHA, which is the substrate for the second step of NOS catalysis (Scheme 1). EPR spectroscopy also reveals a substrate dependence on the spectra [170]. In light of our recent FMN⋯heme distance data [163], these results indicate that the interdomain FMN–heme interactions exert a long-range effect on key heme axial ligand-substrate interactions (Figure 15).…”

Section: Roles Of the Docked Fmn Domain In Tuning Nos Heme Structumentioning

confidence: 54%

“…Ethylene glycol (40%) is added into the sample (as a cryoprotectant) to form a homogenous glassy sample and to minimize spectroscopic diffusion effects. This reagent was also used in low temperature MCD [170, 171] other pulsed EPR [18, 162] studies of NOS proteins.…”

Section: The Interdomain Electron Transfer In Nos Regulationmentioning

confidence: 99%

“…In addition, emerging evidence clearly showed that the docking regulates the NOS catalysis by modulating ferrous heme-NO oxidation reactivity in nNOS [210] and structure [170, 171] of the heme active site in iNOS. Another recent kinetics study also showed that a residue in the FMN domain of iNOS is critical for activity of iNOS [211].…”

Section: Roles Of the Docked Fmn Domain In Tuning Nos Heme Structumentioning

confidence: 99%

“…Moreover, the E→N mutation at E546 in the FMN domain modulates MCD spectra of ferric cyano complexes in a substrate-specific manner [170]. Addition of l -Arg to the wt protein causes notable changes in the CN − -adduct MCD spectrum, while the E546N mutant spectrum is not perturbed (Figure 15).…”

Section: Roles Of the Docked Fmn Domain In Tuning Nos Heme Structumentioning

confidence: 99%

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Mechanism of nitric oxide synthase regulation: Electron transfer and interdomain interactions

Feng

2012

Coordination Chemistry Reviews

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Nitric oxide synthase (NOS), a flavo-hemoprotein, tightly regulates nitric oxide (NO) synthesis and thereby its dual biological activities as a key signaling molecule for vasodilatation and neurotransmission at low concentrations, and also as a defensive cytotoxin at higher concentrations. Three NOS isoforms, iNOS, eNOS and nNOS (inducible, endothelial, and neuronal NOS), achieve their key biological functions by tight regulation of interdomain electron transfer (IET) process via interdomain interactions. In particular, the FMN–heme IET is essential in coupling electron transfer in the reductase domain with NO synthesis in the heme domain by delivery of electrons required for O2 activation at the catalytic heme site. Compelling evidence indicates that calmodulin (CaM) activates NO synthesis in eNOS and nNOS through a conformational change of the FMN domain from its shielded electron-accepting (input) state to a new electron-donating (output) state, and that CaM is also required for proper alignment of the domains. Another exciting recent development in NOS enzymology is the discovery of importance of the the FMN domain motions in modulating reactivity and structure of the catalytic heme active site (in addition to the primary role of controlling the IET processes). In the absence of a structure of full-length NOS, an integrated approach of spectroscopic (e.g. pulsed EPR, MCD, resonance Raman), rapid kinetics (laser flash photolysis and stopped flow) and mutagenesis methods is critical to unravel the molecular details of the interdomain FMN/heme interactions. This is to investigate the roles of dynamic conformational changes of the FMN domain and the docking between the primary functional FMN and heme domains in regulating NOS activity. The recent developments in understanding of mechanisms of the NOS regulation that are driven by the combined approach are the focuses of this review. An improved understanding of the role of interdomain FMN/heme interaction and CaM binding may serve as the basis for the design of new selective inhibitors of NOS isoforms.

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Section: Roles Of the Docked Fmn Domain In Tuning Nos Heme Structumentioning

confidence: 54%

Section: The Interdomain Electron Transfer In Nos Regulationmentioning

confidence: 99%

Section: Roles Of the Docked Fmn Domain In Tuning Nos Heme Structumentioning

confidence: 99%

Section: Roles Of the Docked Fmn Domain In Tuning Nos Heme Structumentioning

confidence: 99%

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Mechanism of nitric oxide synthase regulation: Electron transfer and interdomain interactions

Feng

2012

Coordination Chemistry Reviews

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“…The UV–Vis and MCD spectra of the nNOS proteins were taken in a pH 7.2 buffer (100 mM bis–tris propane, 200 mM NaCl, 40 μM H 4 B, 1 mM DTT, and ~50 % ethylene glycol). Ethylene glycol is added to the protein samples to make an optically transparent glass for MCD measurements (glycerol cannot be used as it causes instability of the protein) [45, 46]. The protein concentration for the MCD samples was typically 25–50 μM, and 1–5 mM L-arginine was added to create the high-spin ferric form of the proteins.…”

Section: Methodsmentioning

confidence: 99%

Exploring second coordination sphere effects in nitric oxide synthase

et al. 2016

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Second coordination sphere (SCS) effects in proteins are modulated by active site residues and include hydrogen bonding, electrostatic/dipole interactions, steric interactions, and π-stacking of aromatic residues. In Cyt P450s, extended H-bonding networks are located around the proximal cysteinate ligand of the heme, referred to as the ‘Cys pocket’. These hydrogen bonding networks are generally believed to regulate the Fe–S interaction. Previous work identified the S(Cys) → Fe σ CT transition in the high-spin (hs) ferric form of Cyt P450cam and corresponding Cys pocket mutants by low-temperature (LT) MCD spectroscopy [Biochemistry 50:1053, 2011]. In this work, we have investigated the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in the hs ferric state (with H4B and L-Arg bound) of rat neuronal nitric oxide synthase oxygenase construct (nNOSoxy) using MCD spectroscopy. For this purpose, wt enzyme and W409 mutants were investigated where the H-bonding network with the axial Cys ligand is perturbed. Overall, the results are similar to Cyt P450cam and show the intense S(Cys) → Fe σ CT band in the LT MCD spectrum at about 27,800 cm−1, indicating that this feature is a hallmark of {heme-thiolate} active sites. The discovery of this MCD feature could constitute a new approach to classify {heme-thiolate} sites in hs ferric proteins. Finally, the W409 mutants show that the hydrogen bond from this group only has a small effect on the Fe–S(Cys) bond strength, at least in the hs ferric form of the protein studied here.

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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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Mutation in the Flavin Mononucleotide Domain Modulates Magnetic Circular Dichroism Spectra of the iNOS Ferric Cyano Complex in a Substrate-Specific Manner

Cited by 12 publications

References 30 publications

Mechanism of nitric oxide synthase regulation: Electron transfer and interdomain interactions

Mechanism of nitric oxide synthase regulation: Electron transfer and interdomain interactions

Exploring second coordination sphere effects in nitric oxide synthase

Elucidating nitric oxide synthase domain interactions by molecular dynamics

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