Buffer optimization for improvement of SpNOXDH thermostability and activity

(A) Thermal unfolding curves of SpNOXDH (0.2 mg.mL−1) in 50mM B bis TRIS-propane buffer pH 6.5, 300 mM NaCl with various glycerol percentages: 5% (–), 10% (–), 15% (–), 20% (–). (B) Thermal unfolding curves of SpNOXDH (0.2 mg.mL−1) in bis TRIS-propane buffer pH 6.5, 5% glycerol with various NaCl concentrations: 100 mM (–), 200 mM (–), 300 mM (–), 400 mM (–), 500 mM (–), 750 mM (–), 1 M (–). (C) Cytochrome c reduction in presence of SpNOXDH (1 µg) NADPH (200 µM) FAD (10 µM) in the standard 50mM bis TRIS-propane buffer at pH 6.5, 300mM NaCl in presence of various glycerol percentages: 0% (–), 5% (–), 10% (–). (D) Cytochrome c reductase activity with SpNOXDH (1 µg), NADPH (200 µM), FAD (10 µM), glycerol (5%), in presence of various NaCl concentrations: 0 mM (–), 100 mM (–), 200 mM (–), 400 mM (–), 1 M (–). (E) 3D surface representation of Tm dependence with NaCl concentrations and glycerol percentages. (○) depicts the optimum used for further experiments of this study.

Effect of cyt. c, FAD, SOD and DPI supplementation on SpNOXDH NADPH oxidase activity

Assays performed with NADPH (200 µM) and SpNOXDH (1µg) in the initial mix. Monitoring of cytochrome c reduction (cyt.c reductase activity) (A) or NADPH oxidation (flavin reductase activity) (B) along with successive addition of FAD and SOD (A and B) or DPI (C) with cyt.c (blue trace) or without (grey trace) in the initial mix. (D) Monitoring simultaneously NADPH oxidation (red) and cytochrome c reduction (blue) in presence of SpNOXDH, cytochrome c, NADPH and FAD, addition of SOD is indicated by an arrow. The table presents the molecular specific activity measured for the different compositions of the initial mixture and addition of the multiple reactants.

FRD containing proteins, represented as surfaces, and accessory proteins (p22phox and DUOXA1) represented as ribbons. TM2 to TM5 were structurally aligned; all proteins are depicted in the same orientation based on this structural alignment. A) Top (extracellular) views of the TM domains of MsrQ (AlfaFold model, green), SpNOX (this work, PDB: 8qq7, gray), TM domain of CsNOX (PDB: 5O0t, purple), NOX2/p22phox (PDB: 8gz3, blue) and DUOX1 (PDB: 7d3f, pink) B). Each protein’s lateral side view (a 90°C rotation from the orientation in A). Black arrow shows potential access to distal heme either from top or from lateral side. Hemes are represented as salmon sticks.

SpNOX and SpNOXDH affinity for flavins. Km and kcat of SpNOX and SpNOXDH for FAD, FMN, riboflavin and lumiflavin. KmDH/Km and kcatDH/kcat indicate the ratio between Kms or kcats measured on the DH domain and on the full-length SpNOX protein. Figure S4 shows the kinetic studies corresponding to the parameters presented here.

Km and Kcat using NADPH or NADH as electron donor for the SpNOX WT, F399W and F397S full length constructs or the corresponding versions of the DH domain (SpNOXDH). F399W is in italics to indicate that position 399 is not homologous to the terminal aromatic in FNR; the W mutation at the homologous position, F397W, did not have sufficient activity to perform such analysis and thus does not appear in this table. WT NADPH/NADH is the ratio of the Km or Kcat for NADPH vs NADH. For SpNOXDH WT Kms were determined by monitoring both Cyt. c reductase activity (at 550 nm) or flavin reductase activity (at 340nm) when reported. The kinetic studies corresponding to the parameters presented here are in Figure S7.

Flavin content and activity measurement of full-length SpNOX WT, SpNox F397S and F397W

A) UV-Visible spectra of SpNOXDH WT, and mutants. Inset) zoom on the 320-500 nm window, a specific spectroscopic feature of the oxidized flavin isoalloxazine ring. B) Reduced cytochrome c was monitored at 550 nm for SpNOX WT (blue), F397W (grey), and F397S (green). A no-protein negative control is shown in black. See also Fig.S6 for spectral characterization of full length SpNOX mutants.

Crystal structures of the DH domain of SpNOX WT and F397W. A) Superposition of SpNOXDH WT (pale cyan ribbon, PDB: 8qq5) and SpNOXDH F397W (light blue ribbon, PDB: 8qq1); FAD is shown as sticks colored by atom, side chains of residues in position 397 (respectively F and W), are shown as sticks in the same color as the corresponding ribbon. B) and C) Superposition of the three different molecules of the asymmetric unit of WT SpNOXDH (B) and of SpNOXDH F397W (C), zoomed on the interaction of the isoalloxazine ring with the aromatic residue at position 397. D) Close up of the FAD binding site in SpNOXDH WT; polar contacts are shown with dotted lines. Colors as for A. E) SpNOXDH with the F397 (pale cyan sticks) and W397 (light blue sticks) residues superimposed; FAD as in A; and NADPH (salmon sticks) shown based on a superposition of the pea FNR:NADPH complex (PDB: 1qfz) with SpNOXDH.

Structure of the full length SpNOX. A) overall structure of SpNOX F397W at 3.6 Å with ribbons in the TM colored by helix, hemes (red sticks) and FAD cofactor (colored by atom). B) The FAD binding site; FAD as in A, side chains (sticks) of residues involved are labeled and interactions are indicated by yellow dotted lines. C) Electron pathway from FAD to distal heme; for clarity, the DH domain and TM1 and TM6 are omitted. Two alternative electron pathways are indicated, from FAD to heme (both depicted as in A), with the Y136 relay between hemes (black dotted lines) or without the relay (red dotted line); distances in Å are indicated.

Determination of Km for flavins as a function of the mutations in the D-loop region. Data here were obtained in the optimal buffer for SpNOX rather than the co-optimized buffer for comparing full-length and DH constructs. It has no impact on flavin Km determination (compare WT line with values from Table2) but Km of full length SpNOX for NAD(P)H are lower by a factor 2 to 3 compared to the co-optimized buffer. For the corresponding curve see figure S10.

Comparison of domain and cofactor organization in NOX2, SpNOX and hDUOX1

A) Proteins were superimposed and are shown in identical orientations based on superposition, resting state NOX2 (PDB: 8gz3, gray ribbon), active SpNOX (this work, PDB: 8qq7, green ribbon) and high Ca2+ DUOX1 in (PDB: 7d3f, red ribbon), with DH domains in darker color. B) and C) DH domains only are represented in same orientations and color as in A. In B, angle and translation from NOX2 to SpNOXDH are indicated. In C, angle and translation from SpNOXDH to DUOX1 are indicated. D) Cofactors necessary for electron transfer are shown as sticks, in identical orientations as in A-C, with distances indicated. In SpNOX the sidechain of Trp397 is shown and the distance to FAD isoalloxazine ring indicated. For DUOX1, NADPH is shown and the distance of nicotinamide to the isoalloxazine indicated.

Comparison of FNR domains and their putative NADPH binding sites

Top, pea FNR (PDB: 1qfz), DUOX1 domain (PDB: 7d3f) in complex with NADPH. Middle, SpNOX DH domain (PDB: 8qq7) and NOX2 DH domain (PDB: 8gz3). Bottom, SpNOX DH is represented with the NADPH from pea FNR after superposition of those proteins; NOX2 DH is represented with the NADPH from DUOX1 after superposition of those proteins. Arg residues conserved between DUOX1 and NOX2 are highlighted using CPK colors, Side chain of R446 is not defined in NOX2 CryoEM structure. In top and middle line, pale yellow and lime patches represent NADPH motifs 1 and 4, respectively (see figureS1). Assuming that NADPH binding implies movement of F397, residues 397FKF399 have been omitted in the SpNOX DH surface to increase clarity and visibility of NAD(P)H binding.