Insights into the nitric oxide reductase mechanism of flavodiiron proteins from a flavin-free enzyme

Abstract

Flavodiiron proteins (FDPs) catalyze reductive scavenging of dioxygen and nitric oxide in air-sensitive microorganisms. FDPs contain a distinctive non-heme diiron/flavin mononucleotide (FMN) active site. Alternative mechanisms for the nitric oxide reductase (NOR) activity consisting of either protonation of a diiron-bridging hyponitrite or "super-reduction" of a diferrous-dinitrosyl by the proximal FMNH(2) in the rate-determining step have been proposed. To test these alternative mechanisms, we examined a deflavinated FDP (deflavo-FDP) from Thermotoga maritima. The deflavo-FDP retains an intact diiron site but does not exhibit multiturnover NOR or O(2) reductase (O(2)R) activity. Reactions of the reduced (diferrous) deflavo-FDP with nitric oxide were examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. Anaerobic addition of nitric oxide up to one NO per diferrous deflavo-FDP results in formation of a diiron-mononitrosyl complex characterized by a broad S = (1)/(2 )EPR signal arising from antiferromagnetic coupling of an S = (3)/(2) {FeNO}(7) with an S = 2 Fe(II). Further addition of NO results in two reaction pathways, one of which produces N(2)O and the diferric site and the other of which produces a stable diiron-dinitrosyl complex. Both NO-treated and as-isolated deflavo-FDPs regain full NOR and O(2)R activities upon simple addition of FMN. The production of N(2)O upon addition of NO to the mononitrosyl deflavo-FDP supports the hyponitrite mechanism, but the concomitant formation of a stable diiron-dinitrosyl complex in the deflavo-FDP is consistent with a super-reduction pathway in the flavinated enzyme. We conclude that a diiron-mononitrosyl complex is an intermediate in the NOR catalytic cycle of FDPs.

Figure 1

Top Panel: homodimeric backbone structure of M. thermoacetica FDP (PDB ID 1YCF) showing iron atoms as red spheres and FMN as orange sticks. Bottom Panel: superposition of the diiron sites in Mt FDP (PDB ID 1YCF) and Tm FDP (PDB ID 1VME). For the Mt FDP diiron site, iron ligand side chains are depicted as CPK-colored sticks, iron as purple spheres, and bridging solvent as a red sphere. All corresponding Tm FDP atoms are colored green. Proximal isoalloxazine ring of FMN in Mt FDP is also shown. Images and superposition were generated in PyMOL (DeLano Scientific LLC).

Figure 2

UV-vis spectra of oxidized (orange), dithionite reduced (black), and reduced deflavo-FDP after addition of 1 equiv (red), 2 equiv (green) and 1 atm NO (blue) at room temperature (protein concentration = 147 μM in 50 mM MOPS pH 7.4; extinction coefficient per diiron site). Also shown is the difference spectrum [1-atm NO spectrum] – 0.65 [oxidized spectrum] (black dashed line).

Figure 3

EPR spectra of oxidized deflavo-FDP (top trace), dithionite-reduced deflavo-FDP (second trace), and reduced deflavo-FDP after the addition of 1 equiv (third trace), 2 equiv (forth trace) and 0.05 atm NO headspace (bottom trace) at 4.2 K. Conditions: protein concentration, 100 μM in 50 mM MOPS pH 7.4; microwave frequency, 9.66 GHz; microwave power, 2 mW; modulation amplitude, 10 G.

Figure 4

Room temperature RR spectra of reduced deflavo-FDP (protein concentration ~ 1 mM in 50 mM MOPS pH 7.4) before (green) and after exposure to excess ¹⁴NO (top black), ¹⁵NO (top red), and ¹⁵N¹⁸O (top blue). The three bottom spectra are “NO complexes” minus “reduced” difference spectra color-coded as for the top spectra. The spectral subtractions were normalized on the 1003-cm⁻¹ band from Phe; the sharp 980-cm⁻¹ band originates from sulfate byproduct of dithionite oxidation.

Figure 5

FTIR difference spectra (“dark” minus “illuminated”) of deflavo-FDP(¹⁴NO)₂ (black) and deflavo-FDP(¹⁵N O)₂ (grey) obtained below 30 K (protein concentration ~ 1.2 mM).

Figure 6

Room temperature RR difference spectra (“NO complexes” minus “reduced protein”) of deflavo-FDP(NO) formed with ¹⁴NO (top trace), ¹⁵NO (middle trace), and ¹⁵N¹⁸O (bottom trace) (protein concentration ~ 1 mM in 50 mM MOPS pH 7.4).

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