Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human endothelial NOS reductase domain

Nishino, Yoshitaka; Yamamoto, Keita; Kimura, Shigenobu; Kikuchi, Akihiro; Shiro, Yoshitsugu; Iyanagi, Takashi

doi:10.1016/j.abb.2007.05.021

Cited by 8 publications

(11 citation statements)

References 61 publications

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“…In addition, the time-dependent increase of flavin fluorescence owing to the release of free FMN (49) is minimized in phosphate buffer. A similar buffer preference in the cytochrome c reduction assay was also reported (50). Therefore, 25 mM potassium phosphate, pH 7.5, 0.1 mM KCl, has been the buffer of the choice for all the steady-state activity assays.…”

Section: Table 2 Reduction Of Fmn By Dithionite In the Fmn Domain Of mentioning

confidence: 77%

Exploring the Electron Transfer Properties of Neuronal Nitric-oxide Synthase by Reversal of the FMN Redox Potential

Das

Sibhatu

et al. 2008

Journal of Biological Chemistry

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In nitric-oxide synthase (NOS) the FMN can exist as the fully oxidized (ox), the one-electron reduced semiquinone (sq), or the two-electron fully reduced hydroquinone (hq). In NOS and microsomal cytochrome P450 reductase the sq/hq redox potential is lower than that of the ox/sq couple, and hence it is the hq form of FMN that delivers electrons to the heme. Like NOS, cytochrome P450BM3 has the FAD/FMN reductase fused to the C-terminal end of the heme domain, but in P450BM3 the ox/sq and sq/hq redox couples are reversed, so it is the sq that transfers electrons to the heme. This difference is due to an extra Gly residue found in the FMN binding loop in NOS compared with P450BM3. We have deleted residue Gly-810 from the FMN binding loop in neuronal NOS (nNOS) to give ⌬G810 so that the shorter binding loop mimics that in cytochrome P450BM3. As expected, the ox/sq redox potential now is lower than the sq/hq couple. ⌬G810 exhibits lower NO synthase activity but normal levels of cytochrome c reductase activity. However, unlike the wild-type enzyme, the cytochrome c reductase activity of ⌬G810 is insensitive to calmodulin binding. In addition, calmodulin binding to ⌬G810 does not result in a large increase in FMN fluorescence as in wild-type nNOS. These results indicate that the FMN domain in ⌬G810 is locked in a unique conformation that is no longer sensitive to calmodulin binding and resembles the "on" output state of the calmodulin-bound wild-type nNOS with respect to the cytochrome c reduction activity.Flavin-containing (FMN or FAD) enzymes catalyze a wide range of reactions and can be classified, according to their functions and reactivity with molecular oxygen, into oxidases, monooxygenases, dehydrogenases, oxidoreductases, and electron transferases (1, 2). The versatility of flavoprotein-catalyzed reactions is attributed to the rich chemistry of the flavin isoalloxazine ring system. Free flavin can exist in three different redox states: oxidized (ox), 3 one-electron reduced semiquinoid (sq), and two-electron reduced hydroquinoid (hq) species (3, 4), as shown in Fig. 1. The semiquinone radical can have two forms depending on whether or not the N5 atom is protonated (5). The anionic semiquinone is red, whereas the neutral semiquinone is blue, each with its own distinct UV-visible absorption features (6). The negative charge on the anionic form of the semiquinone or hydroquinone is localized on the N1-C2ϭO group (3). The redox potentials of the ox/sq and sq/hq couples are Ϫ314 mV and Ϫ124 mV, respectively, for free FMN (7), although the FMN redox potentials and the pK a of N5 can vary dramatically from one flavoprotein to another. It is the variations of flavin-protein interactions in different flavoproteins that give rise to the versatility of flavin redox properties tailored to the specific chemical reaction catalyzed by the particular flavoenzyme (2).Before being identified as a heme-containing enzyme (8 -10), nitric-oxide synthase (NOS) was first recognized as a flavoprotein (11, 12). The C-terminal domain of ra...

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Section: Table 2 Reduction Of Fmn By Dithionite In the Fmn Domain Of mentioning

confidence: 77%

Exploring the Electron Transfer Properties of Neuronal Nitric-oxide Synthase by Reversal of the FMN Redox Potential

Das

Sibhatu

et al. 2008

Journal of Biological Chemistry

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“…Likewise, electron transfer in the reductase domain is also CaM-sensitive in human endothelial NOS (Nishino et al, 2007). Therefore, we hypothesized that the CaM antagonist, DY-9836, would prevent superoxide generation and NO generation during prolonged PE treatment.…”

Section: Discussionmentioning

confidence: 99%

Phenylephrine-Induced Cardiomyocyte Injury Is Triggered by Superoxide Generation through Uncoupled Endothelial Nitric-Oxide Synthase and Ameliorated by 3-[2-[4-(3-Chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxyindazole (DY-9836), a Novel Calmodulin Antagonist

Han

Shioda³

et al. 2008

Mol Pharmacol

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“…Conceivably, all these effects could help determine the catalytic activity of NOSr. In addition, although the properties and electron transfer reactions of nNOSr have been studied in depth (11,28,(31)(32)(33)(34)(35)(36)(37), the amount of data available for eNOSr is much less (20,38,39).…”

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confidence: 99%

Differences in a Conformational Equilibrium Distinguish Catalysis by the Endothelial and Neuronal Nitric-oxide Synthase Flavoproteins

Ilagan

Tiso

Konas

et al. 2008

Journal of Biological Chemistry

153

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Nitric oxide (NO) is a physiological mediator synthesized by NO synthases (NOS).Despite their structural similarity, endothelial NOS (eNOS) has a 6-fold lower NO synthesis activity and 6 -16-fold lower cytochrome c reductase activity than neuronal NOS (nNOS), implying significantly different electron transfer capacities. We utilized purified reductase domain constructs of either enzyme (bovine eNOSr and rat nNOSr) to investigate the following three mechanisms that may control their electron transfer: (i) the set point and control of a two-state conformational equilibrium of their FMN subdomains; (ii) the flavin midpoint reduction potentials; and (iii) the kinetics of NOSr-NADP ؉ interactions. Although eNOSr and nNOSr differed in their NADP(H) interaction and flavin thermodynamics, the differences were minor and unlikely to explain their distinct electron transfer activities. In contrast, calmodulin (CaM)-free eNOSr favored the FMN-shielded (electron-accepting) conformation over the FMN-deshielded (electron-donating) conformation to a much greater extent than did CaM-free nNOSr when the bound FMN cofactor was poised in each of its three possible oxidation states. NADPH binding only stabilized the FMN-shielded conformation of nNOSr, whereas CaM shifted both enzymes toward the FMN-deshielded conformation. Analysis of cytochrome c reduction rates measured within the first catalytic turnover revealed that the rate of conformational change to the FMN-deshielded state differed between eNOSr and nNOSr and was rate-limiting for either CaM-free enzyme. We conclude that the set point and regulation of the FMN conformational equilibrium differ markedly in eNOSr and nNOSr and can explain the lower electron transfer activity of eNOSr.

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Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human endothelial NOS reductase domain

Cited by 8 publications

References 61 publications

Exploring the Electron Transfer Properties of Neuronal Nitric-oxide Synthase by Reversal of the FMN Redox Potential

Exploring the Electron Transfer Properties of Neuronal Nitric-oxide Synthase by Reversal of the FMN Redox Potential

Phenylephrine-Induced Cardiomyocyte Injury Is Triggered by Superoxide Generation through Uncoupled Endothelial Nitric-Oxide Synthase and Ameliorated by 3-[2-[4-(3-Chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxyindazole (DY-9836), a Novel Calmodulin Antagonist

Differences in a Conformational Equilibrium Distinguish Catalysis by the Endothelial and Neuronal Nitric-oxide Synthase Flavoproteins

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