In nitric-oxide synthases (NOSs), two flexible hinges connect the FMN domain to the rest of the enzyme and may guide its interactions with partner domains for electron transfer and catalysis. We investigated the role of the FMN-FAD/NADPH hinge in rat neuronal NOS (nNOS) by constructing mutants that either shortened or lengthened this hinge by 2, 4, and 6 residues. Shortening the hinge progressively inhibited electron flux through the calmodulin (CaM)-free and CaM-bound nNOS to cytochrome c, whereas hinge lengthening relieved repression of electron flux in CaM-free nNOS and had no impact or slowed electron flux through CaM-bound nNOS to cytochrome c. How hinge length influenced heme reduction depended on whether enzyme flavins were pre-reduced with NADPH prior to triggering heme reduction. Without pre-reduction, changing the hinge length was deleterious; with pre-reduction, the hinge shortening was deleterious, and hinge lengthening increased heme reduction rates beyond wild type. Flavin fluorescence and stopped-flow kinetic studies on CaM-bound enzymes suggested hinge lengthening slowed the domain-domain interaction needed for FMN reduction. All hinge length changes lowered NO synthesis activity and increased uncoupled NADPH consumption. We conclude that several aspects of catalysis are sensitive to FMN-FAD/NADPH hinge length and that the native hinge allows a best compromise among the FMN domain interactions and associated electron transfer events to maximize NO synthesis and minimize uncoupled NADPH consumption.
"Moreover, the biphasic nature of the reduction of CaM-free nNOS by NADPH turned out to be also ionic strength dependent and could not therefore be caused by partial degradation or reoxidation of the sample. Using a series of amino acid deletions or insertions in the linker between the FMN and FAD domains, Haque et al. evidenced how the length of this small region controls ET between flavins and between FMN and heme [109,110]. Evidently, the control of ET by the linker length further strengthens the hypotheses of dynamical exchange between the shielded and deshielded forms in NOS. "
[Show abstract][Hide abstract] ABSTRACT: Diflavin reductases are essential proteins capable of splitting the two-electron flux from reduced pyridine nucleotides to a variety of one electron acceptors. The primary sequence of diflavin reductases shows a conserved domain organization harboring two catalytic domains bound to the FAD and FMN flavins sandwiched by one or several non-catalytic domains. The catalytic domains are analogous to existing globular proteins: the FMN domain is analogous to flavodoxins while the FAD domain resembles ferredoxin reductases. The first structural determination of one member of the diflavin reductases family raised some questions about the architecture of the enzyme during catalysis: both FMN and FAD were in perfect position for interflavin transfers but the steric hindrance of the FAD domain rapidly prompted more complex hypotheses on the possible mechanisms for the electron transfer from FMN to external acceptors. Hypotheses of domain reorganization during catalysis in the context of the different members of this family were given by many groups during the past twenty years. This review will address the recent advances in various structural approaches that have highlighted specific dynamic features of diflavin reductases.
International Journal of Molecular Sciences 12/2012; 13(11):15012-41. DOI:10.3390/ijms131115012 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: NADPH-cytochrome P450 reductase is a key component of the P450 mono-oxygenase drug-metabolizing system. There is evidence for a conformational equilibrium involving large-scale domain motions in this enzyme. We now show, using small-angle X-ray scattering (SAXS) and small-angle neutron scattering, that delivery of two electrons to cytochrome P450 reductase leads to a shift in this equilibrium from a compact form, similar to the crystal structure, toward an extended form, while coenzyme binding favors the compact form. We present a model for the extended form of the enzyme based on nuclear magnetic resonance and SAXS data. Using the effects of changes in solution conditions and of site-directed mutagenesis, we demonstrate that the conversion to the extended form leads to an enhanced ability to transfer electrons to cytochrome c. This structural evidence shows that domain motion is linked closely to the individual steps of the catalytic cycle of cytochrome P450 reductase, and we propose a mechanism for this.
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide synthase (NOS), a flavo-hemoprotein, is responsible for biosynthesis of nitric oxide (NO) in mammals. Three NOS isoforms, iNOS, eNOS and nNOS (inducible, endothelial, and neuronal NOS), achieve their biological functions by tight control of interdomain electron transfer (IET) process through interdomain interactions. In particular, the FMN-heme IET is essential in coupling electron transfer in the reductase domain with NO synthesis in the heme domain by delivery of electrons required for O2 activation at the catalytic heme site. Emerging evidence indicates that calmodulin (CaM) activates NO synthesis in eNOS and nNOS by a conformational change of the FMN domain from its shielded electron-accepting (input) state to a new electron-donating (output) state, and that CaM is also required for proper alignment of the FMN and heme domains in the three NOS isoforms. In the absence of a structure of full-length NOS, an integrated approach of spectroscopic, rapid kinetic and mutagenesis methods is required to unravel regulation mechanism of the FMN-heme IET process. This is to investigate the roles of the FMN domain motions and the docking between the primary functional FMN and heme domains in regulating NOS activity. The recent developments in this area that are driven by the combined approach are the focuses of this review. A better understanding of the roles of interdomain FMN/heme interactions and CaM binding may serve as a basis for the rational design of new selective modulators of the NOS enzymes.
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