Structural and mechanistic aspects of flavoproteins: electron transfer through the nitric oxide synthase flavoprotein domain

Abstract

Nitric oxide synthases belong to a family of dual-flavin enzymes that transfer electrons from NAD(P)H to a variety of heme protein acceptors. During catalysis, their FMN subdomain plays a central role by acting as both an electron acceptor (receiving electrons from FAD) and an electron donor, and is thought to undergo large conformational movements and engage in two distinct protein-protein interactions in the process. This minireview summarizes what we know about the many factors regulating nitric oxide synthase flavoprotein domain function, primarily from the viewpoint of how they impact electron input/output and conformational behaviors of the FMN subdomain.

Fig. 1

Domain arrangement and electron flow in the NOS dimer.

Fig. 2

Simplified model of arginine hydroxylation in NOS enzymes. Ferric heme receives an electron from FMNH₂/MNH⁻ enabling oxygen binding and formation of a ferrous dioxygen species. A second electron must be delivered from H₄B to eventually form a high valent iron-oxo species that hydroxylates Arg. The H₄B^+• radical has to be reduced before the next catalytic cycle can proceed.

Fig. 3

(A) Domain organization in NOS and related enzymes. NOS includes regulatory elements that are absent in other closely related proteins. (B) Structure of nNOS flavoprotein domain. The FNR and FMN subdomain are shown in green and yellow, respectively. Regulatory elements (β-finger; AI, autoinhibitory insert; CT, C-terminal tail) are shown in pink. The coenzymes FMN (orange), FAD (dark blue) and NADP⁺ (cyan) are shown as sticks. Modeled fragments, not visible in the crystal structure, are shown in light gray. The visible parts of the hinge element between FMN and FNR subdomains are shown in dark blue. (C) CaM exerts an enhancing effect in three electron-transfer steps.

Fig. 4

Global kinetic model for NOS catalysis. Ferric enzyme reduction (k_r) is rate limiting for the biosynthetic reactions (central linear portion). kcat1 and kcat2 are the conversion rates of the Fe^IIO₂ species to products in the Arg and NOHA reactions, respectively. The ferric heme–NO product complex (Fe^IIINO) can either release NO (k_d) or become reduced (k_r) to a ferrous heme–NO complex (Fe^IINO), which reacts with O₂ (k_ox) to regenerate the ferric enzyme. Adapted from Stuehr et al. [51].

Fig. 5

Model of NOS FMN subdomain function in electron transfer and heme reduction. Electron transfer in NOS can be regarded as a three-state model. Equilibrium A indicates the change between a conformation in which FNR and FMN subdomains are interacting (left) and a conformation where the FMN subdomain is deshielded and available for interaction with electron acceptors such as cytochrome c (center). Equilibrium B indicates the transition from the FMN deshielded conformation to a FMN–NOSoxy domain interacting state. See text for details.

Fig. 6

Model and simulations of cytochrome c reduction by NOS enzymes. (A) Scheme of cytochrome c reduction. The model uses four kinetic rates: dissociation (k₁) and association (k₂) of the FMN and FNR subdomains; FMNH^• reduction rate (k₃) and cytochrome c reduction rate (k₄). For simplicity, k₁ and k₂ are assumed to be independent of the flavin reduction state, k4 is assumed to be much faster than the conformational equilibrium so the backwards rates are negligible, oxidized cytochrome c concentration is constant and in 100-fold excess. (B) Apparent rates of steady-state cytochrome c reduction for different FMNH^• reduction (k₃) values. k_obs values were determined by fitting the apparent change in the concentration of reduced cytochrome c versus time to a straight line. The percentage of deshielding is (k₁/(k₁ + k₂)) × 100. See text for details.

Fig. 7

Correlation between nNOS cytochrome c reductase activity and FMN deshielding. The figure plots relative cytochrome c reductase activities of various CaM-free nNOS flavoproteins and CaM-bound wild-type versus their degree of FMN deshielding. All values are relative to NADPH-bound wild-type enzyme, which was given activity and shielding values of unity. Line is a least squares best fit. Adapted from Tiso et al. [33].

Fig. 8

Complementary charges in the FMN–FNR subdomain interface. The electrostatic potential surfaces of the FMN (left) and FNR (right) subdomains show complementary negative charges in the FMN surface that interact with a positively charged surface patch in the FNR module. Adapted from Panda et al. [90].

Fig. 9

Through-heme model for H₄B radical reduction in NOS. H₄B is 17 Å away from the putative FMN-docking surface. Placing the FMN domain in conformations where Lys423 and Glu762 are in close contact enables feasible distances (9–15 Å) for FMN to heme electron transfer but too long (26–32 Å) for direct FMN to H₄B electron transfer. It is proposed that electron transfer proceeds through heme (dashed line) involving two short-distance (< 15 and 3 Å) electron transfer steps. Adapted from Wei et al. [23].

Scheme 1

Reaction catalyzed by NOS.