Abstract
Neuronal NO synthase (nNOS) consists of a reductase domain that binds FAD, FMN, NADPH, and calmodulin, and an oxygenase domain that binds heme, tetrahydrobiopterin, and the substrate L-arginine. One flavin in resting nNOS exists as an air-stable semiquinone radical. During NO synthesis, electron transfer occurs between the flavins and heme iron. We have characterized the nNOS heme iron and flavin semiquinone radical by electron paramagnetic resonance (EPR) spectroscopy. Under anaerobic conditions, the flavin radical spin relaxation was very slow (8 Hz at 22 K) and was enhanced 13-fold by dissolved dioxygen via spin-spin coupling. The flavin radical, probably the semiquinone FMNH·, was shown by progressive microwave power saturation and EPR saturation recovery under anaerobic conditions to be spin-spin coupled with the heme iron located in the nNOS oxygenase domain. Analysis of an nNOS preparation that was devoid of heme but contained the flavin radical revealed that spin-spin coupling increased the rate of flavin radical relaxation by a factor of 15. The presence of bound substrate (L-arginine) or the substrate analogue N(ω)-nitro-L-arginine methylester (NAME) had no effect on the flavin spin relaxation kinetics. The observed g values of the nNOS heme were 7.68, 4.15, and 1.81 and were unchanged by occupation of the substrate binding site by L-arginine or NAME. The substrate also had no effect on the heme zero-field splitting parameter, D = 5.2 cm-1. Together, the data indicate that the flavin and heme redox centers are positioned near each other in nNOS, consistent with their participating in an interdomain electron transfer. The flavin radical is affected by dissolved oxygen, suggesting that its binding site within the reductase domain is partially exposed to solvent, but is unaffected when substrate binds to the oxygenase domain. Substrate binding also appears to take place outside the first coordination shell of the nNOS heme iron.
| Original language | English |
|---|---|
| Pages (from-to) | 2804-2810 |
| Number of pages | 7 |
| Journal | Biochemistry |
| Volume | 35 |
| Issue number | 8 |
| DOIs | |
| Publication status | Published - Feb 27 1996 |
Fingerprint
ASJC Scopus subject areas
- Biochemistry
Cite this
EPR spectroscopic characterization of neuronal NO synthase. / Galli, Chris; MacArthur, Ryan; Abu-Soud, Husam M.; Clark, Pamela; Stuehr, Dennis J.; Brudvig, Gary W.
In: Biochemistry, Vol. 35, No. 8, 27.02.1996, p. 2804-2810.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - EPR spectroscopic characterization of neuronal NO synthase
AU - Galli, Chris
AU - MacArthur, Ryan
AU - Abu-Soud, Husam M.
AU - Clark, Pamela
AU - Stuehr, Dennis J.
AU - Brudvig, Gary W
PY - 1996/2/27
Y1 - 1996/2/27
N2 - Neuronal NO synthase (nNOS) consists of a reductase domain that binds FAD, FMN, NADPH, and calmodulin, and an oxygenase domain that binds heme, tetrahydrobiopterin, and the substrate L-arginine. One flavin in resting nNOS exists as an air-stable semiquinone radical. During NO synthesis, electron transfer occurs between the flavins and heme iron. We have characterized the nNOS heme iron and flavin semiquinone radical by electron paramagnetic resonance (EPR) spectroscopy. Under anaerobic conditions, the flavin radical spin relaxation was very slow (8 Hz at 22 K) and was enhanced 13-fold by dissolved dioxygen via spin-spin coupling. The flavin radical, probably the semiquinone FMNH·, was shown by progressive microwave power saturation and EPR saturation recovery under anaerobic conditions to be spin-spin coupled with the heme iron located in the nNOS oxygenase domain. Analysis of an nNOS preparation that was devoid of heme but contained the flavin radical revealed that spin-spin coupling increased the rate of flavin radical relaxation by a factor of 15. The presence of bound substrate (L-arginine) or the substrate analogue N(ω)-nitro-L-arginine methylester (NAME) had no effect on the flavin spin relaxation kinetics. The observed g values of the nNOS heme were 7.68, 4.15, and 1.81 and were unchanged by occupation of the substrate binding site by L-arginine or NAME. The substrate also had no effect on the heme zero-field splitting parameter, D = 5.2 cm-1. Together, the data indicate that the flavin and heme redox centers are positioned near each other in nNOS, consistent with their participating in an interdomain electron transfer. The flavin radical is affected by dissolved oxygen, suggesting that its binding site within the reductase domain is partially exposed to solvent, but is unaffected when substrate binds to the oxygenase domain. Substrate binding also appears to take place outside the first coordination shell of the nNOS heme iron.
AB - Neuronal NO synthase (nNOS) consists of a reductase domain that binds FAD, FMN, NADPH, and calmodulin, and an oxygenase domain that binds heme, tetrahydrobiopterin, and the substrate L-arginine. One flavin in resting nNOS exists as an air-stable semiquinone radical. During NO synthesis, electron transfer occurs between the flavins and heme iron. We have characterized the nNOS heme iron and flavin semiquinone radical by electron paramagnetic resonance (EPR) spectroscopy. Under anaerobic conditions, the flavin radical spin relaxation was very slow (8 Hz at 22 K) and was enhanced 13-fold by dissolved dioxygen via spin-spin coupling. The flavin radical, probably the semiquinone FMNH·, was shown by progressive microwave power saturation and EPR saturation recovery under anaerobic conditions to be spin-spin coupled with the heme iron located in the nNOS oxygenase domain. Analysis of an nNOS preparation that was devoid of heme but contained the flavin radical revealed that spin-spin coupling increased the rate of flavin radical relaxation by a factor of 15. The presence of bound substrate (L-arginine) or the substrate analogue N(ω)-nitro-L-arginine methylester (NAME) had no effect on the flavin spin relaxation kinetics. The observed g values of the nNOS heme were 7.68, 4.15, and 1.81 and were unchanged by occupation of the substrate binding site by L-arginine or NAME. The substrate also had no effect on the heme zero-field splitting parameter, D = 5.2 cm-1. Together, the data indicate that the flavin and heme redox centers are positioned near each other in nNOS, consistent with their participating in an interdomain electron transfer. The flavin radical is affected by dissolved oxygen, suggesting that its binding site within the reductase domain is partially exposed to solvent, but is unaffected when substrate binds to the oxygenase domain. Substrate binding also appears to take place outside the first coordination shell of the nNOS heme iron.
UR - http://www.scopus.com/inward/record.url?scp=0029982073&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029982073&partnerID=8YFLogxK
U2 - 10.1021/bi9520444
DO - 10.1021/bi9520444
M3 - Article
C2 - 8611587
AN - SCOPUS:0029982073
VL - 35
SP - 2804
EP - 2810
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 8
ER -