Studies of Iron(III) Porphyrinates Containing Silanethiolate Ligands

The chemistry of several iron­(III) porphyrinates containing silanethiolate ligands is described. The complexes are prepared by protonolysis reactions of silanethiols with the iron­(III) precursors, [Fe­(OMe)­(TPP)] and [Fe­(OH)­(H₂O)­(TMP)] (TPP = dianion of <i>meso-</i>tetraphenylporphine; TMP = dianion of <i>meso-</i>tetramesitylporphine). Each of the compounds has been fully characterized in solution and the solid state. The stability of the silanethiolate complexes versus other iron­(III) porphyrinate complexes containing sulfur-based ligands allows for an examination of their reactivity with several biologically relevant small molecules including H₂S, NO, and 1-methylimidazole. Electrochemically, the silanethiolate complexes display a quasi-reversible one-electron oxidation event at potentials higher than that observed for an analogous arenethiolate complex. The behavior of these complexes versus other sulfur-ligated iron­(III) porphyrinates is discussed

The Nitric Oxide Reductase Mechanism of a Flavo-Diiron Protein: Identification of Active-Site Intermediates and Products

The unique active site of flavo-diiron proteins (FDPs) consists of a nonheme diiron-carboxylate site proximal to a flavin mononucleotide (FMN) cofactor. FDPs serve as the terminal components for reductive scavenging of dioxygen or nitric oxide to combat oxidative or nitrosative stress in bacteria, archaea, and some protozoan parasites. Nitric oxide is reduced to nitrous oxide by the four-electron reduced (FMNH₂–Fe^IIFe^II) active site. In order to clarify the nitric oxide reductase mechanism, we undertook a multispectroscopic presteady-state investigation, including the first Mössbauer spectroscopic characterization of diiron redox intermediates in FDPs. A new transient intermediate was detected and determined to be an antiferromagnetically coupled diferrous-dinitrosyl (<i>S</i> = 0, [{FeNO}⁷]₂) species. This species has an exchange energy, <i>J</i> ≥ 40 cm^–1 (<i>J</i><b>S</b>₁ ° <i><b>S</b></i>₂), which is consistent with a hydroxo or oxo bridge between the two irons. The results show that the nitric oxide reductase reaction proceeds through successive formation of diferrous-mononitrosyl (<i>S</i> = ¹/₂, Fe^II{FeNO}⁷) and the <i>S</i> = 0 diferrous-dinitrosyl species. In the rate-determining process, the diferrous-dinitrosyl converts to diferric (Fe^IIIFe^III) and by inference N₂O. The proximal FMNH₂ then rapidly rereduces the diferric site to diferrous (Fe^IIFe^II), which can undergo a second 2NO → N₂O turnover. This pathway is consistent with previous results on the same deflavinated and flavinated FDP, which detected N₂O as a product (Hayashi Biochemistry 2010, 49, 7040). Our results do <i>not</i> support other proposed mechanisms, which proceed either via “super-reduction” of [{FeNO}⁷]₂ by FMNH₂ or through Fe^II{FeNO}⁷ directly to a diferric-hyponitrite intermediate. The results indicate that an <i>S</i> = 0 [{FeNO}⁷}]₂ complex is a proximal precursor to N–N bond formation and N–O bond cleavage to give N₂O and that this conversion can occur without redox participation of the FMN cofactor

Studies of Iron(III) Porphyrinates Containing Silanethiolate Ligands

The chemistry of several iron­(III) porphyrinates containing silanethiolate ligands is described. The complexes are prepared by protonolysis reactions of silanethiols with the iron­(III) precursors, [Fe­(OMe)­(TPP)] and [Fe­(OH)­(H₂O)­(TMP)] (TPP = dianion of <i>meso-</i>tetraphenylporphine; TMP = dianion of <i>meso-</i>tetramesitylporphine). Each of the compounds has been fully characterized in solution and the solid state. The stability of the silanethiolate complexes versus other iron­(III) porphyrinate complexes containing sulfur-based ligands allows for an examination of their reactivity with several biologically relevant small molecules including H₂S, NO, and 1-methylimidazole. Electrochemically, the silanethiolate complexes display a quasi-reversible one-electron oxidation event at potentials higher than that observed for an analogous arenethiolate complex. The behavior of these complexes versus other sulfur-ligated iron­(III) porphyrinates is discussed

Activity assays of NnlA homologs suggest the natural product N-nitroglycine is degraded by diverse bacteria

Linear nitramines (R–N(R′)NO2; R′ = H or alkyl) are toxic compounds, some with environmental relevance, while others are rare natural product nitramines. One of these natural product nitramines is N-nitroglycine (NNG), which is produced by some Streptomyces strains and exhibits antibiotic activity towards Gram-negative bacteria. An NNG degrading heme enzyme, called NnlA, has recently been discovered in the genome of Variovorax sp. strain JS1663 (Vs NnlA). Evidence is presented that NnlA and therefore, NNG degradation activity is widespread. To achieve this objective, we characterized and tested the NNG degradation activity of five Vs NnlA homologs originating from bacteria spanning several classes and isolated from geographically distinct locations. E. coli transformants containing all five homologs converted NNG to nitrite. Four of these five homologs were isolated and characterized. Each isolated homolog exhibited similar oligomerization and heme occupancy as Vs NnlA. Reduction of this heme was shown to be required for NnlA activity in each homolog, and each homolog degraded NNG to glyoxylate, NO2− and NH4+ in accordance with observations of Vs NnlA. It was also shown that NnlA cannot degrade the NNG analog 2-nitroaminoethanol. The combined data strongly suggest that NnlA enzymes specifically degrade NNG and are found in diverse bacteria and environments. These results imply that NNG is also produced in diverse environments and NnlA may act as a detoxification enzyme to protect bacteria from exposure to NNG

A Diferrous-Dinitrosyl Intermediate in the N₂O‑Generating Pathway of a Deflavinated Flavo-Diiron Protein

Flavo-diiron proteins (FDPs) function as anaerobic nitric oxide scavengers in some microorganisms, catalyzing reduction of nitric to nitrous oxide. The FDP from <i>Thermotoga maritima</i> can be prepared in a deflavinated form with an intact diferric site (deflavo-FDP). Hayashi et al. [(2010) <i>Biochemistry 49</i>, 7040–7049] reported that reaction of NO with reduced deflavo-FDP produced substoichiometric N₂O. Here we report a multispectroscopic approach to identify the iron species in the reactions of deflavo-FDP with NO. Mössbauer spectroscopy identified two distinct ferrous species after reduction of the antiferromagnetically coupled diferric site. Approximately 60% of the total ferrous iron was assigned to a diferrous species associated with the N₂O-generating pathway. This pathway proceeds through successive diferrous-mononitrosyl (<i>S</i> = ¹/₂ Fe^II{FeNO}⁷) and diferrous-dinitrosyl (<i>S</i> = 0 [{FeNO}⁷]₂) species that form within ∼100 ms of mixing of the reduced protein with NO. The diferrous-dinitrosyl intermediate converted to an antiferromagnetically coupled diferric species that was spectroscopically indistinguishable from that in the starting deflavinated protein. These diiron species closely resembled those reported for the flavinated FDP [Caranto et al. (2014) <i>J. Am. Chem. Soc</i>. <i>136</i>, 7981–7992], and the time scales of their formation and decay were consistent with the steady state turnover of the flavinated protein. The remaining ∼40% of ferrous iron was inactive in N₂O generation but reversibly bound NO to give an <i>S</i> = ³/₂ {FeNO}⁷ species. The results demonstrate that N₂O formation in FDPs can occur via conversion of <i>S</i> = 0 [{FeNO}⁷]₂ to a diferric form without participation of the flavin cofactor

Vibrational Analysis of Mononitrosyl Complexes in Hemerythrin and Flavodiiron Proteins: Relevance to Detoxifying NO Reductase

Flavodiiron proteins (FDPs) play important roles in the microbial nitrosative stress response in low-oxygen environments by reductively scavenging nitric oxide (NO). Recently, we showed that FMN-free diferrous FDP from <i>Thermotoga maritima</i> exposed to 1 equiv NO forms a stable diiron-mononitrosyl complex (deflavo-FDP­(NO)) that can react further with NO to form N₂O [Hayashi, T.; Caranto, J. D.; Wampler, D. A; Kurtz, D. M., Jr.; Moënne-Loccoz, P. Biochemistry 2010, 49, 7040−7049]. Here we report resonance Raman and low-temperature photolysis FTIR data that better define the structure of this diiron-mononitrosyl complex. We first validate this approach using the stable diiron-mononitrosyl complex of hemerythrin, Hr­(NO), for which we observe a ν­(NO) at 1658 cm^–1, the lowest ν­(NO) ever reported for a nonheme {FeNO}⁷ species. Both deflavo-FDP­(NO) and the mononitrosyl adduct of the flavinated FPD (FDP­(NO)) show ν­(NO) at 1681 cm^–1, which is also unusually low. These results indicate that, in Hr­(NO) and FDP­(NO), the coordinated NO is exceptionally electron rich, more closely approaching the Fe­(III)­(NO^–) resonance structure. In the case of Hr­(NO), this polarization may be promoted by steric enforcement of an unusually small FeNO angle, while in FDP­(NO), the Fe­(III)­(NO^–) structure may be due to a semibridging electrostatic interaction with the second Fe­(II) ion. In Hr­(NO), accessibility and steric constraints prevent further reaction of the diiron-mononitrosyl complex with NO, whereas in FDP­(NO) the increased nucleophilicity of the nitrosyl group may promote attack by a second NO to produce N₂O. This latter scenario is supported by theoretical modeling [Blomberg, L. M.; Blomberg, M. R.; Siegbahn, P. E. J. Biol. Inorg. Chem. 2007, 12, 79−89]. Published vibrational data on bioengineered models of denitrifying heme-nonheme NO reductases [Hayashi, T.; Miner, K. D.; Yeung, N.; Lin, Y.-W.; Lu, Y.; Moënne-Loccoz, P. Biochemistry 2011, 50, 5939−5947] support a similar mode of activation of a heme {FeNO}⁷ species by the nearby nonheme Fe­(II)

<i>Treponema denticola</i> Superoxide Reductase: In Vivo Role, in Vitro Reactivities, and a Novel [Fe(Cys)₄] Site

In vitro and in vivo results are presented demonstrating that superoxide reductase (SOR) from the air-sensitive oral spirochete, <i>Treponema denticola</i> (Td), is a principal enzymatic scavenger of superoxide in this organism. This SOR contains the characteristic non-heme [Fe­(His)₄Cys] active sites. No other metal-binding domain has been annotated for Td SOR. However, we found that Td SOR also accommodates a [Fe­(Cys)₄] site whose spectroscopic and redox properties resemble those in so-called 2Fe-SORs. Spectroscopic comparisons of the wild type and engineered Cys → Ser variants indicate that three of the Cys ligands correspond to those in [Fe­(Cys)₄] sites of “canonical” 2Fe-SORs, whereas the fourth Cys ligand residue has no counterpart in canonical 2Fe-SORs or in any other known [Fe­(Cys)₄] protein. Structural modeling is consistent with iron ligation of the “noncanonical” Cys residue across subunit interfaces of the Td SOR homodimer. The Td SOR was isolated with only a small percentage of [Fe­(Cys)₄] sites. However, quantitative formation of stable [Fe­(Cys)₄] sites was readily achieved by exposing the as-isolated protein to an iron salt, a disulfide reducing agent and air. The disulfide/dithiol status and iron occupancy of the Td SOR [Fe­(Cys)₄] sites could, thus, reflect intracellular redox status, particularly during periods of oxidative stress