A docked state conformational dynamics model to explain the ionic strength dependence of FMN – heme electron transfer in nitric oxide synthase

Abstract

The FMN-heme interdomain electron transfer (IET) in nitric oxide synthase (NOS) is a key stage of the electron transport chain, which supplies the catalytic heme site(s) with the NADPH-derived electrons. While there is a recognition that this IET depends on both the electron tunneling and the conformational dynamics, the detailed mechanism remains unclear. In this work, the IET kinetics were measured by laser flash photolysis for a bidomain oxygenase/FMN (oxyFMN) construct of human inducible NOS (iNOS) over the ionic strength range from 0.1 to 0.5 M. The forward (heme → FMN, k) and backward (FMN → heme, k) intrinsic IET rate constants were determined from the analysis of the observed IET rates using the additional information regarding the conformational dynamics obtained from the FMN fluorescence lifetime measurements and theoretical estimates. Both k and k exhibit a bell-shaped dependence on the ionic strength, I, with the maximum rates corresponding to I ~ 0.2 M. This dependence was explained using a new model, which considers the effect of formation of pairs between the protein surface charged residues and solution ions on the docked state dynamics. The trial simulations of the intrinsic IET rate dependences using this model show that the data can be reproduced using reasonable energetic, structural, and chemical parameters. The suggested model can explain both the monophasic and biphasic ionic strength dependences and can be used to rationalize the interprotein/interdomain electron transfer rates for other types of protein systems where the docked state is sufficiently long-lived.

Heat shock protein 90 enhances the electron transfer between the FMN and heme cofactors in neuronal nitric oxide synthase

“…Also note that the small but noticeable increase in the IET rate upon Hsp90 complexing is functionally relevant because similar extent of stimulation in nNOS activity by Hsp90 was reported [12]. The rates of chemical reactions at the NOS heme active site are generally comparable with that of the IET [21,22], and the FMN–heme IET thus plays a major role in determining the overall NO production rate. Hence, Hsp90 appears to play an auxiliary but meaningful role in enhancing the essential FMN–heme IET step in nNOS.…”

“…This IET is essential in the delivery of electrons required for O 2 activation and the subsequent NO synthesis in the heme domain [6]. The rates of catalytic reactions at the NOS heme site are generally comparable to that of the IET [21,22]. The FMN–heme IET thus plays a substantial role in the NOS function, which justifies our study of the effect of Hsp90 on the IET kinetics.…”

Effect of Macromolecular Crowding on the FMN–Heme Intraprotein Electron Transfer in Inducible NO Synthase

“…The geometries of these conformational states may be altered in the crowded environment. The FMN domain docked at the heme domain is believed to go through short-range motions to productively dock to the heme domain . This short-range exploration for the optimal docking position (i.e., conformational sampling) results in productive docking of the FMN domain with the heme domain.…”

Generation and characterization of functional phosphoserine-incorporated neuronal nitric oxide synthase holoenzyme

“…An interesting question relates to why is the FMN -heme IET in pSer1412 nNOS protein is slower than wt. The observed IET rate is significantly affected by the conformational dynamics that determine the formation and dissociation of the docking complex between the FMN and heme domains [41,42], yet much remains unknown regarding the mechanistic roles of NOS phosphorylations in the conformational transitions tied to discrete electron transfer steps. S1412 is located at the CT of rat nNOS.…”