Surface Charge Interactions of the FMN Module Govern Catalysis by Nitric-oxide Synthase

Abstract

The FMN module of nitric-oxide synthase (NOS) plays a pivotal role by transferring NADPH-derived electrons to the enzyme heme for use in oxygen activation. The process may involve a swinging mechanism in which the same face of the FMN module accepts and provides electrons during catalysis. Crystal structure shows that this face of the FMN module is electronegative, whereas the complementary interacting surface is electropositive, implying that charge interactions enable function. We used site-directed mutagenesis to investigate the roles of six electronegative surface residues of the FMN module in electron transfer and catalysis in neuronal NOS. Results are interpreted in light of crystal structures of NOS and related flavoproteins. Neutralizing or reversing the negative charge of each residue altered the NO synthesis, NADPH oxidase, and cytochrome c reductase activities of neuronal NOS and also altered heme reduction. The largest effects occurred at the NOS-specific charged residue Glu 762. Together, the results suggest that electrostatic interactions of the FMN module help to regulate electron transfer and to minimize flavin autoxidation and the generation of reactive oxygen species during NOS catalysis.

Surface Charges and Regulation of FMN to Heme Electron Transfer in Nitric-oxide Synthase

“…A cluster of negatively charged residues on the surface of the FMN domain is apparent in the crystal structure of the nNOS reductase domain (19). Mutation of these negatively charged residues inhibited electron transfer between FMN and heme in some cases (20). These results, together with structural data, supported a model for domain interaction where the cluster of negative charges on the FMN domain may interact with a positively charged region on the surface of the nNOSoxy domain (18,19) (see Fig.…”

“…A, to allow NO synthesis, the FMN domain has to shift from a position adequate to accept electrons from FAD (left, FMNshielded conformation) to a conformation in which is able to reach the heme domain (right, FMN-deshielded conformation). Available evidence indicates that the FMN domain uses the same face to interact with FAD and heme domains (20). The signs indicate the charges on each surface.…”

“…Steady-state Assays-Cytochrome c reductase activity was determined as reported previously (20) by following the absorbance change for the reduction of cytochrome c by nNOS at 550 nm using an extinction coefficient of 21 Heme Reduction-The electron transfer from the reductase domain to the heme was studied at 10°C as described previously (20, 30) using a Hi-Tech SF-61 stopped-flow instrument with a diode array detector (Hi-Tech Scientific, Salisbury, UK) equipped for anaerobic work. A protein solution containing 10 M nNOS, 100 mM EPPS, pH 7.6, 100 mM NaCl, 10 M H 4 B, 2 mM L-Arg, 0.5 mM DTT, 50 M CaM, 5 mM CaCl 2 , and 1 mM EDTA, at least half-CO-saturated, was mixed with an anaerobic, CO-saturated solution containing 100 mM EPPS, pH 7.6, 100 mM NaCl, and 100 M NADPH.…”

Molecular architecture of mammalian nitric oxide synthases

“…To transition into the output state, a large conformational change occurs that allows the FMN subdomain to donate an electron to the oxidase domain. Although progress has been made elucidating the interaction surfaces present in the input and output states (7)(8)(9)(10)(11)(12), the trajectory of this conformational change between the input and output is only beginning to be explored (13). Further understanding of this conformational change is particularly important as electron transfer is rate-limiting during NOS catalysis (6).…”

Regulation of Interdomain Interactions by Calmodulin in Inducible Nitric-oxide Synthase

“…CaM regulates NOS activity by controlling the rates of electron transfer between the two flavin cofactors and between FMN and heme (13,(17)(18)(19)(20)(21)(22)(23). The mechanism by which CaM regulates the electron flux in NOS isozymes has been under intensive investigation (19, 24 -26), and recent studies have focused on the movement of the FMN domain and its interactions with the FAD domain and the oxygenase domain in NOS (13,14,16,27) and in other diflavin enzymes (15,28). In addition, there are three elements in the reductase domain of constitutive NOSs that are influenced by the CaM binding: the autoregulatory region (AR, a ϳ40 residue insert in the FMN domain), the C-terminal extension, and, to a lesser extent, the ␤-finger (or SI, a small insert in the FAD/connecting domain) (13, 29 -31), all of which are lacking in iNOS.…”