Oral 1832-2 – Charge Pairing and Phosphorylation Regulate The Conformational Equilibrium and Switching Rates in Neuronal and Endothelial NO Synthase Flavoprotein Domains

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Nitric Oxide


Electron flux through nitric oxide synthase reductase (NOSr) is thought to depend on conformational switching motions of their FMN domains, which enables the enzymes to cycle between closed unreactive and open reactive conformational states. However, the conformational equilibrium setpoints (Keq), rates of conformational switching, and interflavin electron transfer rates are mostly unknown, and how these parameters may combine to determine catalytic activities in NOSs is not well understood. To address these, we determined and compared the conformational equilibrium setpoints and rates of conformational switching between reactive open and unreactive closed states, in wild-type nNOSr and four FMN surface mutants (E762R, E762N, E816R, E819R) of nNOSr, and in wild-type eNOSr and the phospho-mimetic S1179D eNOSr mutant. We used stopped flow spectroscopy, single turnover methods, and a kinetic model that relates conformational setpoint and rates of conformational switching to the electron flux through each enzyme to cytochrome c. We found that charge neutralization or reversal at each of these residues alters the setpoint (Keq) of the NOSr conformational equilibrium to favor the open reactive (FMN-deshielded) conformational state. Moreover, computer simulations of the kinetic traces of cytochrome c reduction by the nNOSr mutants suggest that they have higher conformational transition rates (1.5–4-fold) relative to wild-type nNOSr. Wild-type eNOSr mostly exists in closed conformational state (88% closed, 12% open, Keq = 0.125) with a very slow electron flux. In comparison, the S1179D mutation alters the eNOSr setpoint to Keq = 1.5 (40% closed, 60% open), indicating that the open reactive conformation is favored in S1179D eNOSr. Our computer simulation data suggest that S1179D eNOSr also has a faster conformational transition, and a 20-fold faster opening rate relative to wild-type eNOSr. Thus, mutating Ser1179 to Asp alters both the setpoint and transition rates of equilibrium, and these can fully explain the increased electron flux seen in S1179D eNOSr mutant. Together, our studies provide the first measures of conformational equilibrium settings and conformational switching rates in nNOSr and eNOSr proteins, reveal that remarkable differences exist between the two proteins, and show how charge pairing interactions at the domain interface, or phosphorylation at Ser1179, alter NOS activity by modifying these conformational parameters.