Detecting and understanding the roles of nitric oxide in biology

Abstract

We are pursuing a dual strategy for investigating the chemistry of nitric oxide as a biological signaling agent. In one approach, metal-based fluorescent sensors for the detection of NO in living cells are evaluated, and a sensor based on a copper fluorescein complex has proved to be a valuable lead compound. Sensors of this class permit identification of NO from both inducible and constitutive forms of nitric oxide synthase and facilitate investigation of different NO functions in response to external stimuli. In the other approach, we employ synthetic model complexes of iron-sulfur clusters to probe their reactivity toward nitric oxide as biomimics of the active sites of iron-sulfur proteins. Our studies reveal that NO disassembles the Fe-S clusters to form dinitrosyl iron complexes.

Figure 1

(a) CuFL1 detection of NO in SK-N-SH neuroblastoma cells. [CuFL1] = 1 μM, [17β-estradiol] = 100 nM. From left to right: 25 min exposure to CuFL1, no 17β-estradiol; 5 min; 10 min; 15 min; 25 min exposure to CuFL1 and 17β-estradiol. Top: Fluorescence images; Bottom: DIC images. Scale bars = 50 μm. (b) CuFL1 detection of NO in Raw 264.7 murine macrophages. [CuFL1] = 1 μM, [lipopolysaccharide (LPS)] = 500 ng/mL, [interferon-γ (IFN-γ)] = 250 U/mL. From left to right: 12 h exposure to CuFL1, no LPS/IFN-γ; 6 h; 8 h; 10 h; 12 h exposure to CuFL1 and LPS/IFN-γ. Top: Fluorescence images; Bottom: DIC images. Scale bars = 50 μm. For additional details see ref.

Figure 2

(a) Relative energies of the molecular orbitals for the ground states of FL1 (left) and FL1-NO (right). (b) Qualitative molecular orbital diagram for the ground (left), excited (middle), and charge-transfer (right) states of FL1. R1 = 2-methylquinoline; R2 = fluorophore. For additional details see ref. .

Figure 3

Visualization of fluorescence enhancement by CuFL1 in J774A.1 infected macrophages at 2 and 18 h post-infection. Top row: bNOS-expressing B. anthracis (Sterne) cells taken up by macrophages. Sterne cells produce NO using bNOS within 2 h of uptake. The host macrophages produce NO using iNOS between 2 and 18 h. Middle row: bNOS-deficient B. anthracis cells taken up by macrophages. No fluorescence is observed after 2 h because these bacteria cannot generate NO. The host macrophages produce NO using iNOS as usual. Bottom row: Control, no bacteria and no induction of iNOS

Figure 4

Structure of human glutathione S-transferase P1-1 monomer containing a bound dinitrosyl iron complex. Coordinates were taken from PDB code 1ZGN; for additional details see ref. .

Figure 5

Cyclic voltammogram of (Et₄N)[Fe(NO)₂(BIPM)] displaying the reversible {Fe(NO)₂}^9/10 couple. Conditions: 3 mM in CH₃CN; glassy carbon electrode; 0.1 M Bu₄NPF₆ electrolyte; 100 mV/s scan rate.

Figure 6

Comparison of selected spectroscopic and structural properties for two homologous DNIC redox partners. Thermal ellipsoids are drawn at 50% probability. Hydrogen atoms and the PPN⁺ cation in the {Fe(NO)₂} structure are omitted for clarity. For additional details see ref. .

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Chart 1

Common {Fe(NO₂)}-containing species.