scholarly journals Charge-pairing interactions control the conformational setpoint and motions of the FMN domain in neuronal nitric oxide synthase

2013 ◽  
Vol 450 (3) ◽  
pp. 607-617 ◽  
Author(s):  
Mohammad Mahfuzul Haque ◽  
Mekki Bayachou ◽  
Mohammed A. Fadlalla ◽  
Deborah Durra ◽  
Dennis J. Stuehr

The NOS (nitric oxide synthase; EC 1.14.13.39) enzymes contain a C-terminal flavoprotein domain [NOSred (reductase domain of NOS)] that binds FAD and FMN, and an N-terminal oxygenase domain that binds haem. Evidence suggests that the FMN-binding domain undergoes large conformational motions to shuttle electrons between the NADPH/FAD-binding domain [FNR (ferredoxin NADP-reductase)] and the oxygenase domain. Previously we have shown that three residues on the FMN domain (Glu762, Glu816 and Glu819) that make charge-pairing interactions with the FNR help to slow electron flux through nNOSred (neuronal NOSred). In the present study, we show that charge neutralization or reversal at each of these residues alters the setpoint [Keq(A)] of the NOSred conformational equilibrium to favour the open (FMN-deshielded) conformational state. Moreover, computer simulations of the kinetic traces of cytochrome c reduction by the mutants suggest that they have higher conformational transition rates (1.5–4-fold) and rates of interflavin electron transfer (1.5–2-fold) relative to wild-type nNOSred. We conclude that the three charge-pairing residues on the FMN domain govern electron flux through nNOSred by stabilizing its closed (FMN-shielded) conformational state and by retarding the rate of conformational switching between its open and closed conformations.

2001 ◽  
Vol 276 (40) ◽  
pp. 37506-37513 ◽  
Author(s):  
Jian Zhang ◽  
Pavel Martàsek ◽  
Rosemary Paschke ◽  
Thomas Shea ◽  
Bettie Sue Siler Masters ◽  
...  

2004 ◽  
Vol 279 (19) ◽  
pp. 19824-19831 ◽  
Author(s):  
Stéphane Marchal ◽  
Antonius C. F. Gorren ◽  
Morten Sørlie ◽  
K. Kristoffer Andersson ◽  
Bernd Mayer ◽  
...  

Oxygen binding to the oxygenase domain of reduced endothelial nitric oxide synthase (eNOS) results in two distinct species differing in their Soret and visible absorbance maxima and in their capacity to exchange oxygen by CO. At 7 °C, heme-oxy I (with maxima at 420 and 560 nm) is formed very rapidly (kon≈ 2.5·106m–1·s–1) in the absence of substrate but in the presence of pterin cofactor. It is capable of exchanging oxygen with CO at –30 °C. Heme-oxy II is formed more slowly (kon≈ 3·105m–1·s–1) in the presence of substrate, regardless of the presence of pterin. It is also formed in the absence of both substrate and pterin. In contrast to heme-oxy I, it cannot exchange oxygen with CO at cryogenic temperature. In the presence of arginine, heme-oxy II is characterized by absorbance maxima near 432, 564, and 597 nm. When arginine is replaced byN-hydroxyarginine, and also in the absence of both substrate and pterin, its absorbance maxima are blue-shifted to 428, 560, and 593 nm. Heme-oxy I seems to resemble the ferrous dioxygen complex observed in many hemoproteins, including cytochrome P450. Heme-oxy II, which is the oxygen complex competent for product formation, appears to represent a distinct conformation in which the electronic configuration is essentially locked in the ferric superoxide complex.


2001 ◽  
Vol 360 (1) ◽  
pp. 247 ◽  
Author(s):  
Hirohito YONEYAMA ◽  
Akira YAMAMOTO ◽  
Hiroaki KOSAKA

Sign in / Sign up

Export Citation Format

Share Document