Control of electron transfer in neuronal NO synthase

Abstract

T h e nitric oxide synthases (NOSs) are dimeric flavocytochromes consisting of an oxygenase domain with cytochrome P450-like Cys-ligated haem, coupled to a diflavin reductase domain, which is related to cytochrome P450 reductase. T h e NOSs catalyse the sequential mono-oxygenation of arginine to N-hydroxyarginine and then to citrulline and NO. The constitutive NOS isoforms (cNOSs) are regulated by calmodulin (CaM), which binds at elevated concentrations of free Ca2+, whereas the inducible isoform binds CaM irreversibly. One of the main structural differences between the constitutive and inducible isoforms is an insert of 40-50 amino acids in the FMN-binding domain of the cNOSs. Deletion of the insert in rat neuronal NOS (nNOS) led to a mutant enzyme which binds CaM at lower Ca2+ concentrations and which retains activity in the absence of CaM. In order to resolve the mechanism of action of CaM activation we determined reduction potentials for the F M N and FAD cofactors of rat nNOS in the presence and absence of CaM using a recombinant form of the reductase domain. T h e results indicate that CaM binding does not modulate the reduction potentials of the flavins, but appears to control electron transfer primarily via a large structural rearrangement. We

NO Formation by Neuronal NO‐Synthase can be Controlled by Ultrafast Electron Injection from a Nanotrigger

“…Electron flow was estimated by determining the ratio (R) of the forward electron transfer rate (k ET ) to the observed decay (by charge recombination as in Equation (4) in CaM (À) or by formation of FADH8 via Equations (3) and (4) in CaM (+)). CaM binding increased this ratio by a factor of 6.2, from R = 3.7 to 23 ( Table 2).…”

“…[1][2] Calmodulin (CaM) binding at high Ca II concentrations mediates electron transfer (ET) flow between the heme (subunit B) and reductase domains (subunit A). [4][5] Cal-A C H T U N G T R E N N U N G modulin binding is thought to induce structural rearrangements required for catalysis and is an absolute requisite for NO formation from constitutive NOS, endothelial NOS (eNOS), and neuronal NOS (nNOS). [6][7] These enzymes generate the low concentrations of NO used in signaling pathways to regulate blood flow and pressure, neuronal development, and neurotransmission.…”

Control of Electron Transfer and Catalysis in Neuronal Nitric-oxide Synthase (nNOS) by a Hinge Connecting Its FMN and FAD-NADPH Domains

“…We know that the two-electron reduced nNOS flavoprotein domain can formally exist in three states as follows: FADH 2 /FMN, the di-semiquinone FADH/FMNH, and FAD/FMNH 2 . The flavin midpoint couples in nNOS are poised to favor a blend of the 2nd and 3rd states (21,51,52), and populating the 3rd state enables a second hydride transfer from NADPH to FAD in the second phase of flavin reduction. Because the hinge extensions are unlikely to impact the flavin midpoint potentials, we assume that the flavin midpoint potentials in the hinge mutants remain similar to wild type.…”

Solving Kinetic Equations for the Laser Flash Photolysis Experiment on Nitric Oxide Synthases: Effect of Conformational Dynamics on the Interdomain Electron Transfer

“…The interdomain electron transfer (IET) processes represent key steps in NOS catalysis. ,, They are regulated by calmodulin (CaM), which binds to the CaM-binding region of the linker joining the FMN and heme domains. Although the CaM binding to NOS has little to no effect on the thermodynamics of redox processes, − it facilitates the IET from the FAD hydroquinone to FMN semiquinone (FMNH • ) within the reductase domain and enables the IET from the FMN hydroquinone (FMN hq ) to the catalytic heme iron in the heme domain . This indicates that the NOS regulation by CaM is accomplished dynamically through controlling conformational changes required for effective IET. − …”