Electron transfer in nitric-oxide synthase

Sagami, Ikuko; Sato, Yuko; Noguchi, Tomoko; Miyajima, Michihito; Rozhkova, Elena A.; Daff, Simon; Shimizu, Tôru

doi:10.1016/s0010-8545(01)00446-5

Cited by 11 publications

(9 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…An Enzymatic Study. ± Nitric monoxide (NO) is a relatively stable radical and a very unique signaling molecule in terms of its mode of interaction with targets involving electron transfer [47], as well as the breadth of resulting biological responses, including relaxation of vascular tissue [48], inhibition of platelet aggregation [49] [50], and central and/or peripheral neural signal transductions [51]. NO is produced from l-arginine in the body by the action of NO synthase (NOS) [52].…”

Section: Methodsmentioning

confidence: 99%

Chemical and Biological Investigations of β‐Oligoarginines

Seebàch¹,

Namoto²,

Mahajan³

et al. 2004

Chemistry & Biodiversity

View full text Add to dashboard Cite

In view of the important role arginine plays in living organisms as the free amino acid and, especially, as a residue in peptides and proteins, the homologous beta-homoarginines are central in our investigations of beta-peptides (Fig. 1). The preparation of beta2-homoarginine derivatives suitably protected for solution- or solid-phase peptide syntheses is described with full experimental detail (9 and 12 in Scheme 1). The readily available Fmoc-beta3 hArg(Boc)2-OH is used for manual solid-phase synthesis of beta3-oligoarginines (on Rink amide or Rink amide AM resin) either by single amino acid coupling (Scheme 3) or, much better, by dimer-fragment coupling (Scheme 4). In this way, beta3-oligoarginine amides composed of 4, 6, 7, 8, and 10 residues, both with and without fluorescein labelling, were synthesized (Schemes 2-4), purified by preparative HPLC and identified by high-resolution mass spectrometry. The free amino acids (R)- and (S)-H-beta2 hArg-OH and (S)-H-beta3 hArg-OH were tested for their ability to function as substrates for NO synthase (iNOS); the beta3-oligoarginine amides (5, 6, and 7 residues) were tested for antibacterial (against six pathogens) and hemolytic (against rat and human erythrocytes) activities. All test results were negative: none of the free beta-homoarginines induced NO formation (Fig. 3), and there was no lysis of erythrocytes (concentrations up to 100 microM; Table 1), and no significant antibiotic activity (MIC > or = 64 microg/ml; Table 2). Cell-penetration studies with the fluorescence-labelled, peptidase-resistant beta3-oligoarginine amides were carried out with HeLa cells and human foreskin keratinocytes (HFKs). The results obtained with fluorescence microscopy are: i) the longer-chain beta-oligoarginine amides (8 and 10 residues; Figs. 4-6) enter the cells and end up in the nuclei, especially in the nucleoli, irrespective of temperature (37 degrees and 4 degrees with HFKs) or pretreatment with NaN3 (with HFKs), indicating a non-endocytotic and non-energy-dependent uptake mechanism; ii) the beta-tetraarginine derivative occupies the cell surface but does not enter the cells (with HeLa); iii) the cell-growth rate of the HFKs is not affected by a 1-microM concentration of the fluorescence-labelled beta-octaarginine amide (Fig. 7), i.e., there is no antiproliferative effect. In vivo experiments with mouse skin and the beta-octaarginine derivative show migration of the beta-peptide throughout the epidermis (Fig. 8). As a contribution to understanding the mechanism, we have also studied the behavior of fluorescence-labelled beta-octa- and beta-decaarginine amides (TFA salts) towards giant unilamellar vesicles (GUVs) built of neutral (POPC) or anionic (POPC/POPG mixtures) phospholipids: the beta-oligoarginine amides bind tightly to the surface of anionic GUVs but do not penetrate the lipid bilayer (Fig. 9) as they do with living cells. In contrast, a beta-heptapeptide FL-22, which had been used as a negative control sample for the cell-penetration experiments, entered the GUVs of ne...

show abstract

Section: Methodsmentioning

confidence: 99%

Chemical and Biological Investigations of β‐Oligoarginines

Seebàch¹,

Namoto²,

Mahajan³

et al. 2004

Chemistry & Biodiversity

View full text Add to dashboard Cite

show abstract

“…Although many details of the NOS mechanism remain to be elucidated, it is well-established that IET processes are key steps in the regulation of NO synthesis (16,17). It is thus of significant interest to investigate the thermodynamics (18)(19)(20) and kinetics (13,14,21) of electron transfer (ET).…”

mentioning

confidence: 99%

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

et al. 2006

View full text Add to dashboard Cite

Intersubunit intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation, which serves as the input state for reduction of FMN by electrons from NADPH and flavin adenine dinucleotide (FAD) in the reductase domain. To favor the formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct of rat neuronal NOS (nNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of IET between the FMN and heme domains in the nNOS oxyFMN construct in the presence and absence of added calmodulin (CaM) were directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single-domain heme oxygenase constructs. The IET rate constant in the presence of CaM (262 s -1 ) was increased approximately 10-fold compared to that in the absence of CaM (22 s -1 ). The effect of CaM on interdomain interactions was further evidenced by electron paramagnetic resonance (EPR) spectra. This work provides the first direct evidence of the CaM control of electron transfer (ET) between FMN and heme domains through facilitation of the FMN/heme interactions in the output state. Therefore, CaM controls IET between heme and FMN domains by a conformational gated mechanism. This is essential in coupling ET in the reductase domain in NOS with NO synthesis in the oxygenase domain.

show abstract

“…Chapon et al rovněž popsali rozklad přirozených barviv piva, vína i barevného indikátoru methylové červeni v anaerobním prostředí za účasti peroxidu vodíku a kyseliny askorbové, tuto reakci však pokládali za typickou radikálovou oxidaci, ačkoliv lze v literatuře nalézt příklady oxidačního i redukčního degradačního mechanismu [13,15,16]. V každém případě tato reakce představuje příklad degradačního působení za nepřítomnosti kyslíku.…”

Section: úVodunclassified

Aerobic and Anaerobic Beer Ageing.

Šavel¹

2003

Kvasny Prum

View full text Add to dashboard Cite

Electron transfer in nitric-oxide synthase

Cited by 11 publications

References 39 publications

Chemical and Biological Investigations of β‐Oligoarginines

Chemical and Biological Investigations of β‐Oligoarginines

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

Aerobic and Anaerobic Beer Ageing.

Contact Info

Product

Resources

About