Spectroscopy and DFT Calculations of a Flavo-diiron Enzyme Implicate New Diiron Site Structures

Abstract

Flavo-diiron proteins (FDPs) are non-heme iron containing enzymes that are widespread in anaerobic bacteria, archaea, and protozoa, serving as the terminal components to dioxygen and nitric oxide reductive scavenging pathways in these organisms. FDPs contain a dinuclear iron active site similar to that in hemerythrin, ribonucleotide reductase, and methane monooxygenase, all of which can bind NO and O2. However, only FDP competently turns over NO to N2O. Here, EPR and Mössbauer spectroscopies allow electronic characterization of the diferric and diferrous species of FDP. The exchange-coupling constant J (Hex = JS1·S2) was found to increase from +20 cm−1 to +32 cm−1 upon reduction of the diferric to the diferrous species, indicative of (1) at least one hydroxo bridge between the iron ions for both states and (2) a change to the diiron core structure upon reduction. In comparison to characterized diiron proteins and synthetic complexes, the experimental values were consistent with a dihydroxo bridged diferric core, which loses one hydroxo bridge upon reduction. DFT calculations of these structures gave values of J and Mössbauer parameters in agreement with experiment. Although the crystal structure shows a hydrogen bond between the iron bound aspartate and the bridging solvent molecule, the DFT calculations of structures consistent with the crystal structure gave calculated values of J incompatible with the spectroscopic results. We conclude that the crystal structure of the diferric state does not represent the frozen solution structure and that a mono-μ-hydroxo diferrous species is the catalytically functional state that reacts with NO and O2. The new EPR spectroscopic probe of the diferric state indicated that the diferric structure of FDP prior to and immediately after turnover with NO are flavin mononucleotide (FMN) dependent, implicating an additional proton transfer role for FMN in turnover of NO.

Spectroscopy and DFT Calculations of Flavo–Diiron Nitric Oxide Reductase Identify Bridging Structures of NO-Coordinated Diiron Intermediates

“…The fitted exchange-coupling constant J = 17(2) cm −1 is consistent with a hydroxo bridge between Fe P and Fe D . 12 The fitting of the data used minimal assumptions, specifically, g FeNO = 2.0, (E/D) FeNO ≈ 0, values which are typical of all known nonheme {FeNO} 7 protein sites, and the values for g x and g y of the Fe 2+ site determined from eq 2. For comparable magnitudes of J and D, as in the case of FDP NO , the observed S = ½ g-tensor depends on J and the D - and g -tensors of the individual iron centers.…”

“…Oxo- and aqua-bridged diiron sites are precluded as their exchange couplings are, respectively, an order of magnitude larger and smaller than the experimental value. 12…”

“…28,29 First and second coordination sphere atoms were included and optimized as described previously (see Supporting Information). 12 The FDP crystal structures show that the FMN cofactor has no direct contact with any of the iron ligands, and because there is no change in the FMNH 2 oxidation state among FDP red , FDP NO , and FDP diNO , 12 the FMN was excluded from the DFT models. The exchange-coupling constants between paramagnetic subunits A and B were calculated with the expression J AB = 2(E f − E BS )/n A n B , where n A and n B are the numbers of unpaired electrons associated with A and B, respectively, and E F and E BS are the energies for the ferromagnetic state (F) and broken-symmetry configuration (BS).…”

“…30–32 The reliability of the calculated J values using our DFT methodology was previously verified for a number of synthetic diferric and diferrous complexes. 12 Details of the DFT treatment of the exchange interactions involving the paramagnetic nitrosyl ligands in FDP NO and FDP diNO are given in the Supporting Information. The calculations for the 57 Fe isomer shifts (δ) and quadrupole splitting (ΔE q ) were calibrated as described previously.…”

“…As shown in Figure 1, our previous spectroscopic characterization of FMN-diferric FDP (FDP ox ) from T. maritima (Tm) identified a bis-μ-hydroxo diferric structure that was inconsistent with the crystal structure. 12 This inconsistency may be specific to Tm FDP or due to differences between frozen solution and the crystalline state. The spectroscopic studies also showed that the FMNH 2 -diferrous state of Tm FDP (FDP red ) contains a mono-μ-hydroxo diiron structure shown in Figure 1 but without Fe D -coordinated water (O w ).…”