Control of eNOS and nNOS through regulation of obligatory conformational changes in the reductase catalytic cycle

Abstract

Endothelial and neuronal nitric oxide synthases (eNOS, nNOS) are important signal generators in a number of processes including angiogenesis and neurotransmission. The homologous inducible isoform (iNOS) occupies a multitude of conformational states in a catalytic cycle, including subnanosecond input and output states and a distribution of 'open' conformations with average lifetimes of ~4.3 ns. In this study, fluorescence lifetime spectroscopy was used to probe conformational states of purified eNOS and nNOS in the presence of chaotropes, calmodulin, NADP + and NADPH. Two-domain FMN/oxygenase constructs of nNOS were also examined with respect to calmodulin effects. Optical biosensing was used to analyze calmodulin binding in the presence of NADP + and NADPH. Calmodulin binding induced a shift of the population away from the input and to the open and output states of NOS. NADP + shifted the population towards the input state. The oxygenase domain, lacking the input state, provided a measure of calmodulin-induced open-output transitions. A mechanism for regulation by calmodulin and an elucidation of the catalytic mechanism are suggested by a 'conformational lockdown' model. Calmodulin speeds transitions between input and open and between open and output states, effectively reducing the conformational manifold, speeding catalysis. Conformational control of catalysis involves reorientation of the FMN binding domain, of which fluorescence lifetime is an indicator. The approach described herein is a new tool for biophysical and structural analysis of NOS enzymes, regulatory events and other homologous reductase-containing enzymes. 3 A note to the readerThis manuscript has over the past several years been submitted to and rejected by several journals, usually on the basis of reviewer opinion that it was not an important enough result to merit inclusion in the journal. Owing to the passing of the first author and the loss of his expertise in fluorescence lifetime spectroscopy, it has become too onerous a task to continually revise the manuscript to suit the whims of reviewers who nevertheless still reject the work. We are thus simply releasing the final form of the manuscript to BioRxiv in the hopes that it finds a readership who will find, as we do, that the results are of value to the field.

Nitroxyl: A Novel Strategy to Circumvent Diabetes Associated Impairments in Nitric Oxide Signaling

“…This occurs through its vasodilator capacity, inhibition and reversal of platelet aggregation, suppression of inflammation and oxidative stress, inhibition of thrombosis and modulation of vascular smooth muscle cell (VSMC) proliferation and vascular remodeling (Napoli et al, 2006;Ignarro, 2019). NO• is endogenously synthesized by three isoforms of the NO• synthase (NOS) enzyme, specifically, neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS), also known as NOS1, NOS2 and NOS3, respectively (Salerno et al, 2018). All three enzymes consist of two subunits, an N-terminal oxygenase domain that binds the substrate L-arginine, cofactor tetrahydrobiopterin (BH 4 ) and a heme iron group, and a Cterminal reductase domain that binds nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide phosphate (FAD) and flavin mononucleotide (FMN) (Qian and Fulton, 2013).…”