Predicted Structure of Oxygenase (NOSoxy) and Reductase (NOSred) Domains from O. tauri NOS.
(A) and (B) Ribbon diagram of Ot NOSoxy (A) generated according to the coordinates of the crystallized eNOSoxy of B. taurus (B). Inset: Magnified region showing the catalytic site of NOS, which contains heme, H₄B, and l-Arg.
(C) Superposition of residues in the active site of Ot NOS (green) and eNOS (red). Hydrogen bonds are indicated as dotted lines.
(D) Structural alignment between model of Ot NOSred (green) and nNOSred from R. norvegicus (red). Ot NOS contains a shorter CD2A loop and lacks the ACE segment. Note that the nNOS structure represented lacks part of the ACE segment. Due to the high motility of the ACE segment, it was not modeled; therefore, residues Ser-849 to Gly-872 are not depicted. The first and last residues Ser-849 and Gly-872 of the crystal structure of nNOS are shown in ball and stick in light green.

Purification and Spectral Characteristics of Recombinant NOS from O. tauri Expressed in E. coli.
(A) SDS-PAGE analysis of NOS during different steps of purification. Lane 1, cell extracts of E. coli transformed with the empty pET24b vector and induced with IPTG; lane 2, extracts of E. coli transformed with pET24b-OtNOS and induced with IPTG; lane 3, eluted fraction from 2′,5′-ADP-agarose loaded with protein extracts from E. coli pET24b-OtNOS. MW, molecular weight standards.
(B) Immunoblot analysis of the eluted fraction from 2′,5′-ADP-agarose using a specific anti-OtNOS antibody.
(C) Absolute absorbance spectra of recombinant NOS. Solid black line, unperturbed spectrum; dashed green line, after the addition of 1 mM imidazole; dotted red line, after the addition of 1 mM imidazole and 200 μM l-Arg. Experiments were performed using 0.5 μg purified NOS.
[See online article for color version of this figure.]

NO Generation by O. tauri.
(A) NO fluorescence emitted by O. tauri cells (grown at 100 μmol m⁻² s⁻¹ light irradiance) was determined using the probe DAF-FM DA in the presence of different concentrations of l-Arg. Data are expressed as fold increase (arbitrary units per min) with respect to the control. Inset: Representative graph of three independent experiments. Arrow indicates the point used for calculations of NO production. Asterisks indicate a statistically significant difference (t test, P < 0.05). Error bars denote se (n = 3).
(B) NO fluorescence was determined in an O. tauri cell culture treated with 5 mM l-Arg in the presence and absence of the NOS inhibitors, l-NAME (10 mM), l-NMMA (10 mM), and l-NNA (10 mM), the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) (20 μM), or the CaM antagonist TFP (100 μM). Data are expressed as fold increase (arbitrary units per min) with respect to the control. Different letters indicate a statistically significant difference (ANOVA, P < 0.05). Error bars denote se (n = 3).
(C) O. tauri cells treated or not with 5 mM l-Arg for 20 min in the presence of DAF-FM DA. NO fluorescence was visualized using a fluorescence microscope. Bars = 50 μm.

Alignment of O. tauri NOS (Ot NOS) and Human NOS Sequences.
(A) The sequences of Ot NOS and human eNOS, nNOS, and iNOS were aligned using ClustalX and Genedoc software. Black boxes indicate conserved residues in all four sequences, dark-gray boxes represent conserved residues in three sequences, and light-gray boxes represent conserved residues in two sequences. Amino acids that share no similarity are unshaded. Putative cofactor binding sites for zinc, Heme, H₄B, CaM, FMN, FAD pyrophosphate, FAD isoalloxazine, NADPH ribose, NADPH adenine, and the C-terminal domain of NADPH are shown. Regions involved in the dimerization interface (i.e., regions I, II, III, and IV) are boxed.
(B) Comparison of the domain architecture of NOS from different sources. Eukaryotic NOS enzymes have a Zn binding region, a NOSoxy domain that binds heme, l-Arg, and H₄B, a CaM binding region, and a NOSred domain with subdomains that bind FMN, FAD, and NAD. Bacterial NOS enzymes contain only a NOSoxy domain. The question mark indicates that the Zn binding motif is not completely conserved. H₄B/THF means that is not clear whether NOS-containing bacteria synthesize H₄B, although they can use tetrahydrofolate (THF) as a pterin cofactor (Crane et al., 2010).

Phylogenetic Tree of NOS Sequences.
Radial representation of consensus maximum likelihood tree obtained from 1000 replicates using the JTT+F+Gamma model. Colors indicate main taxonomic divisions: vertebrates (red), invertebrates (blue), plants (green), bacteria (yellow), and amoebozoa (light blue). The accession number of each sequence is given next to the species name. Values in the branches indicate bootstrap percentage. Bootstrap values below 40% are not shown.

NO Production and Peroxide Sensitivity in E. coli Expressing Recombinant O. tauri NOS.
(A) E. coli culture was induced with IPTG in the presence or absence of 1 mM l-Arg. NO fluorescence was determined in a fluorometer using the DAF-FM DA probe. Data are expressed as fold increase of arbitrary units per min with respect to the control (E. coli transformed with the empty vector pET24b). A representative graph of three independent experiments is shown.
(B) IPTG-induced E. coli cultures (expressing O. tauri NOS or harboring an empty pET24b vector) were treated with 1 mM l-Arg or d-Arg. Fluorescence was determined in the presence of DAF-FM DA over a 2-h period. Data expressed as fold increase with respect to the control. Different letters indicate statistically significant differences (ANOVA, P < 0.05). Error bars denote se (n = 3).
(C) E. coli cultures were treated with 30 mM H₂O₂ for 30 min in the presence of 1 mM l-Arg. Cell death was determined using the Sytox Green probe. Values are expressed as fold increase with respect to the control. The asterisk indicates statistically significant difference between the H₂O₂ treatment and the control (t test, P < 0.05). Error bars denote se (n = 5).
(D) NO content measured with the Griess reagent. Different letters indicate a statistically significant difference (ANOVA, P < 0.05). Error bars denote se (n = 3).

NO Generation Is Light Irradiance and Growth Phase Dependent.
(A) O. tauri cells were grown at 40 μmol m⁻² s⁻¹ light irradiance. Culture growth was determined by measuring the OD at 660 nm. NO production was determined using DAF-FM DA with or without the addition of 5 mM l-Arg. Asterisks indicate a statistically significant difference (t test, P < 0.05). Error bars denote se (n = 3). AU, arbitrary units; DW, dry weight.
(B) O. tauri cells grown at 40 μmol m⁻² s⁻¹ of light irradiance for 10 d were transferred to 100 μmol m⁻² s⁻¹ for 24 h. NO production was determined in the presence or absence of 5 mM l-Arg using DAF-FM DA. Data are expressed as fold increase (arbitrary units per min) with respect to the control. Asterisks indicate a statistically significant difference (t test, P < 0.05). Error bars denote se (n = 3).
(C) Immunoblot analysis of NOS expression in O. tauri cultured for 2, 5, and 10 d at 40 μmol m⁻² s⁻¹ light irradiance or 7 d at 40 μmol m⁻² s⁻¹ and shift to 100 μmol m⁻² s⁻¹ for another 3 d (7 → 10). In another experiment, cells were grown at 100 μmol m⁻² s⁻¹ light irradiance for 10 d and treated with or without 5 mM l-Arg for 1 h. Ot NOS protein was detected with a commercial anti-iNOS antibody. The ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RbcL) stained with Ponceau was included as a protein loading control. Relative quantification of Ot NOS and RbcL from blots using Image J software is shown in the bar graph.
(D) O. tauri cells grown at 40 μmol m⁻² s⁻¹ were transferred to 400 μmol m⁻² s⁻¹ for 1 h and then returned to 40 μmol m⁻² s⁻¹ for 18 h. NO production was assayed using the fluorescent probe DAF-FM DA. Data are expressed as fold increase (arbitrary units per min) with respect to cells grown at 40 μmol m⁻²s ⁻¹. Different letters indicate a statistically significant difference (ANOVA, P < 0.05). Error bars denote se (n = 3).