Vibrational analyses of the reaction of oxymyoglobin with NO using a photolabile caged NO donor at cryogenic temperatures.

The NO dioxygenation reaction catalyzed by heme-containing globin proteins is a crucial aerobic detoxification pathway. Accordingly, the second order reaction of NO with oxymyoglobin and oxyhemoglobin has been the focus of a large number of kinetic and spectroscopic studies. Stopped-flow and rapid-freeze-quench (RFQ) measurements have provided evidence for the formation of a Fe(III)-nitrato complex with millisecond lifetime prior to release of the nitrate product, but the temporal resolution of these techniques is insufficient for the characterization of precursor species. Most mechanistic models assume the formation of an initial Fe(III)-peroxynitrite species prior to homolytic cleavage of the OO bond and recombination of the resulting NO2 and Fe(IV)=O species. Here we report vibrational spectroscopy measurements for the reaction of oxymyoglobin with a photolabile caged NO donor at cryogenic temperatures. We show that this approach offers efficient formation and trapping of the Fe(III)-nitrato, enzyme-product, complex at 180 K. Resonance Raman spectra of the Fe(III)-nitrato complex trapped via RFQ in the liquid phase and photolabile NO release at cryogenic temperatures are indistinguishable, demonstrating the complementarity of these approaches. Caged NO is released by irradiation <180 K but diffusion into the heme pocket is fully inhibited. Our data provide no evidence for Fe(III)-peroxynitrite of Fe(IV)=O species, supporting low activation energies for the NO to nitrate conversion at the oxymyoglobin reaction site. Photorelease of NO at cryogenic temperatures allows monitoring of the reaction by transmittance FTIR which provides valuable quantitative information and promising prospects for the detection of protein sidechain reorganization events in NO-reacting metalloenzymes.

Reductive Coupling of Nitric Oxide by Cu(I): Stepwise Formation of Mono- and Dinitrosyl Species En Route to a Cupric Hyponitrite Intermediate.

Transition-metal-mediated reductive coupling of nitric oxide (NO(g)) to nitrous oxide (N2O(g)) has significance across the fields of industrial chemistry, biochemistry, medicine, and environmental health. Herein, we elucidate a density functional theory (DFT)-supplemented mechanism of NO(g) reductive coupling at a copper-ion center, [(tmpa)CuI(MeCN)]+ (1) {tmpa = tris(2-pyridylmethyl)amine}. At -110 °C in EtOH (<-90 °C in MeOH), exposing 1 to NO(g) leads to a new binuclear hyponitrite intermediate [{(tmpa)CuII}2(µ-N2O22-)]2+ (2), exhibiting temperature-dependent irreversible isomerization to the previously characterized κ2-O,O'-trans-[(tmpa)2Cu2II(µ-N2O22-)]2+ (OOXray) complex. Complementary stopped-flow kinetic analysis of the reaction in MeOH reveals an initial mononitrosyl species [(tmpa)Cu(NO)]+ (1-(NO)) that binds a second NO molecule, forming a dinitrosyl species [(tmpa)CuII(NO)2] (1-(NO)2). The decay of 1-(NO)2 requires an available starting complex 1 to form a dicopper-dinitrosyl species hypothesized to be [{(tmpa)Cu}2(µ-NO)2]2+ (D) bearing a diamond-core motif, en route to the formation of hyponitrite intermediate 2. In contrast, exposing 1 to NO(g) in 2-MeTHF/THF (v/v 4:1) at <-80 °C leads to the newly observed transient metastable dinitrosyl species [(tmpa)CuII(NO)2] (1-(NO)2) prior to its disproportionation-mediated transformation to the nitrite product [(tmpa)CuII(NO2)]+. Our study furnishes a near-complete profile of NO(g) activation at a reduced Cu site with tripodal tetradentate ligation in two distinctly different solvents, aided by detailed spectroscopic characterization of metastable intermediates, including resonance Raman characterization of the new dinitrosyl and hyponitrite species detected.

Oxygen versus Sulfur Coordination in Cobalt Superoxo Complexes: Spectroscopic Properties, O2 Binding, and H-Atom Abstraction Reactivity.

A five-coordinate, disiloxide-ligated cobalt(II) (S = 3/2) complex (1) was prepared as an oxygen-ligated analogue to the previously reported silanedithiolate-ligated CoII(Me3TACN)(S2SiMe2) (J. Am. Chem. Soc., 2019, 141, 3641-3653). The structural and spectroscopic properties of 1 were analyzed by single-crystal X-ray diffraction, electron paramagnetic resonance (EPR), and NMR spectroscopies. The reactivity of 1 with dioxygen was examined, and it was shown to bind O2 reversibly in a range of solvents at low temperatures. A cobalt(III)-superoxo complex, CoIII(O2·-)(Me3TACN)((OSi2Ph)2O) (2), was generated, and was analyzed by UV-vis, EPR, and resonance Raman spectroscopies. Unlike its sulfur-ligated analogue, complex 2 can thermally release O2 to regenerate 1. Vibrational assignments for selective 18O isotopic labeling of both O2 and disiloxide ligands in 2 are consistent with a 6-coordinate, Co(η1-O2·-)("end-on") complex. Complex 2 reacts with the O-H bond of 4-methoxy-2,2,6,6-tetramethylpiperidin-1-ol (4-MeO-TEMPOH) via H-atom abstraction with a rate of 0.58(2) M-1 s-1 at -105 °C, but it is unable to oxidize phenol substrates. This bracketed reactivity suggests that the O-H bond being formed in the putative CoIII(OOH) product has a relatively weak O-H bond strength (BDFE ∼66-74 kcal mol-1). These thermodynamic and kinetic parameters are similar to those seen for the sulfur-ligated Co(O2)(Me3TACN)(S2SiMe2), indicating that the differences in the electronic structure for O versus S ligation do not have a large impact on H-atom abstraction reactivity.

Influence of the Interdomain Interface on Structural and Redox Properties of Multiheme Proteins.

Multiheme proteins are important in energy conversion and biogeochemical cycles of nitrogen and sulfur. A diheme cytochrome c4 (c4) was used as a model to elucidate roles of the interdomain interface on properties of iron centers in its hemes A and B. Isolated monoheme domains c4-A and c4-B, together with the full-length diheme c4 and its Met-to-His ligand variants, were characterized by a variety of spectroscopic and stability measurements. In both isolated domains, the heme iron is Met/His-ligated at pH 5.0, as in the full-length c4, but becomes His/His-ligated in c4-B at higher pH. Intradomain contacts in c4-A are minimally affected by the separation of c4-A and c4-B domains, and isolated c4-A is folded. In contrast, the isolated c4-B is partially unfolded, and the interface with c4-A guides folding of this domain. The c4-A and c4-B domains have the propensity to interact even without the polypeptide linker. Thermodynamic cycles have revealed properties of monomeric folded isolated domains, suggesting that ferrous (FeII), but not ferric (FeIII) c4-A and c4-B, is stabilized by the interface. This study illustrates the effects of the interface on tuning structural and redox properties of multiheme proteins and enriches our understanding of redox-dependent complexation.

Direct Reduction of NO to N2O by a Mononuclear Nonheme Thiolate Ligated Iron(II) Complex via Formation of a Metastable {FeNO}7 Complex.

Addition of NO to a nonheme dithiolate-ligated iron(II) complex, FeII(Me3TACN)(S2SiMe2) (1), results in the generation of N2O. Low-temperature spectroscopic studies reveal a metastable six-coordinate {FeNO}7 intermediate (S = 3/2) that was trapped at -135 °C and was characterized by low-temperature UV-vis, resonance Raman, EPR, Mössbauer, XAS, and DFT studies. Thermal decay of the {FeNO}7 species leads to the evolution of N2O, providing a rare example of a mononuclear thiolate-ligated {FeNO}7 that mediates NO reduction to N2O without the requirement of any exogenous electron or proton sources.

Spectroscopic Characterization of a Diferric Mycobacterial Hemerythrin-Like Protein with Unprecedented Reactivity toward Nitric Oxide.

Hemerythrin-like proteins (HLPs) are broadly distributed across taxonomic groups and appear to play highly diverse functional roles in prokaryotes. Mycobacterial HLPs contribute to the survival of these pathogenic bacteria in mammalian macrophages, but their modes of action remain unclear. A recent crystallographic characterization of Mycobacterium kansasii HLP (Mka-HLP) revealed the unexpected presence of a tyrosine sidechain (Tyr54) near the coordination sphere of one of the two iron centers. Here, we show that Tyr54 is a true ligand to the Fe2(III) ion which, in conjunction with the presence of a µ-oxo group bridging the two iron(III), brings unique reactivity toward nitric oxide (NO). Monitoring the titration of Mka-HLP with NO by Fourier-transform infrared and electron paramagnetic resonance spectroscopies shows that both diferric and diferrous forms of Mka-HLP accumulate an uncoupled high-spin and low-spin {FeNO}7 pair. We assign the reactivity of the diferric protein to an initial radical reaction between NO and the µ-oxo bridge to form nitrite and a mixed-valent diiron center that can react further with NO. Amperometric measurements of NO consumption by Mka-HLP confirm that this reactivity can proceed at low micromolar concentrations of NO, before additional NO consumption, supporting a NO scavenging role for mycobacterial HLPs.

An Iron(III) Superoxide Corrole from Iron(II) and Dioxygen.

A new structurally characterized ferrous corrole [FeII (ttppc)]- (1) binds one equivalent of dioxygen to form [FeIII (O2-. )(ttppc)]- (2). This complex exhibits a 16/18 O2 -isotope sensitive ν(O-O) stretch at 1128 cm-1 concomitantly with a single ν(Fe-O2 ) at 555 cm-1 , indicating it is an η1 -superoxo ("end-on") iron(III) complex. Complex 2 is the first well characterized Fe-O2 corrole, and mediates the following biologically relevant oxidation reactions: dioxygenation of an indole derivative, and H-atom abstraction from an activated O-H bond.

A Reactive, Photogenerated High-Spin (S = 2) FeIV(O) Complex via O2 Activation.

Addition of dioxygen at low temperature to the non-heme ferrous complex FeII(Me3TACN)((OSiPh2)2O) (1) in 2-MeTHF produces a peroxo-bridged diferric complex Fe2III(µ-O2)(Me3TACN)2((OSiPh2)2O)2 (2), which was characterized by UV-vis, resonance Raman, and variable field Mössbauer spectroscopies. Illumination of a frozen solution of 2 in THF with white light leads to homolytic O-O bond cleavage and generation of a FeIV(O) complex 4 (ν(Fe=O) = 818 cm-1; δ = 0.22 mm s-1, ΔEQ = 0.23 mm s-1). Variable field Mössbauer spectroscopy measurements show that 4 is a rare example of a high-spin S = 2 FeIV(O) complex and the first synthetic example to be generated directly from O2. Complex 4 is highly reactive, as expected for a high-spin ferryl, and decays rapidly in fluid solution at cryogenic temperatures. This decay process in 2-MeTHF involves C-H cleavage of the solvent. However, the controlled photolysis of 2 in situ with visible light and excess phenol substrate leads to competitive phenol oxidation, via the proposed transient generation of 4 as the active oxidant.

A Nonheme Mononuclear {FeNO}7 Complex that Produces N2 O in the Absence of an Exogenous Reductant.

A new nonheme iron(II) complex, FeII (Me3 TACN)((OSiPh2 )2 O) (1), is reported. Reaction of 1 with NO(g) gives a stable mononitrosyl complex Fe(NO)(Me3 TACN)((OSiPh2 )2 O) (2), which was characterized by Mössbauer (δ=0.52 mm s-1 , |ΔEQ |=0.80 mm s-1 ), EPR (S=3/2), resonance Raman (RR) and Fe K-edge X-ray absorption spectroscopies. The data show that 2 is an {FeNO}7 complex with an S=3/2 spin ground state. The RR spectrum (λexc =458 nm) of 2 combined with isotopic labeling (15 N, 18 O) reveals ν(N-O)=1680 cm-1 , which is highly activated, and is a nearly identical match to that seen for the reactive mononitrosyl intermediate in the nonheme iron enzyme FDPnor (ν(NO)=1681 cm-1 ). Complex 2 reacts rapidly with H2 O in THF to produce the N-N coupled product N2 O, providing the first example of a mononuclear nonheme iron complex that is capable of converting NO to N2 O in the absence of an exogenous reductant.

Axial Heme Coordination by the Tyr-His Motif in the Extracellular Hemophore HasAp Is Critical for the Release of Heme to the HasR Receptor of Pseudomonas aeruginosa.

Pseudomonas aeruginosa senses extracellular heme via an extra cytoplasmic function σ factor that is activated upon interaction of the hemophore holo-HasAp with the HasR receptor. Herein, we show Y75H holo-HasAp interacts with HasR but is unable to release heme for signaling and uptake. To understand this inhibition, we undertook a spectroscopic characterization of Y75H holo-HasAp by resonance Raman (RR), electron paramagnetic resonance (EPR), and X-ray crystallography. The RR spectra are consistent with a mixed six-coordinate high-spin (6cHS), six-coordinate low-spin (6cLS) heme configuration and an H218O exchangeable FeIII-O stretching frequency with 16O/18O and H/D isotope shifts that support a two-body Fe-OH2 oscillator with (iron-hydroxy)-like character as both hydrogen atoms are engaged in short hydrogen bond interactions with protein side chains. Further support comes from the EPR spectrum of Y75H holo-HasAp that shows a LS rhombic signal with ligand-field splitting values intermediate between those of His-hydroxy and bis-His ferric hemes. The crystal structure of Y75H holo-HasAp confirmed the coordinated solvent molecule hydrogen bonded through H75 and H83. The long-range conformational rearrangement of HasAp upon heme binding can still take place in Y75H holo-HasAp, because the intercalation of a hydroxy ligand between the heme iron and H75 allows the variant to reproduce the heme binding pocket observed in wild-type holo-HasAp. However, in the absence of a covalent linkage to the Y75 loop combined with the malleability provided by the bracketing H75 and H83 hydrogen bonds, either the hydroxy sixth ligand remains bound after complexation of Y75H holo-HasAp with HasR or rearrangement and coordination of H85 prevent heme transfer.

Stepwise nitrosylation of the nonheme iron site in an engineered azurin and a molecular basis for nitric oxide signaling mediated by nonheme iron proteins.

Mononitrosyl and dinitrosyl iron species, such as {FeNO}7, {FeNO}8 and {Fe(NO)2}9, have been proposed to play pivotal roles in the nitrosylation processes of nonheme iron centers in biological systems. Despite their importance, it has been difficult to capture and characterize them in the same scaffold of either native enzymes or their synthetic analogs due to the distinct structural requirements of the three species, using redox reagents compatible with biomolecules under physiological conditions. Here, we report the realization of stepwise nitrosylation of a mononuclear nonheme iron site in an engineered azurin under such conditions. Through tuning the number of nitric oxide equivalents and reaction time, controlled formation of {FeNO}7 and {Fe(NO)2}9 species was achieved, and the elusive {FeNO}8 species was inferred by EPR spectroscopy and observed by Mössbauer spectroscopy, with complemental evidence for the conversion of {FeNO}7 to {Fe(NO)2}9 species by UV-Vis, resonance Raman and FT-IR spectroscopies. The entire pathway of the nitrosylation process, Fe(ii) → {FeNO}7 → {FeNO}8 → {Fe(NO)2}9, has been elucidated within the same protein scaffold based on spectroscopic characterization and DFT calculations. These results not only enhance the understanding of the dinitrosyl iron complex formation process, but also shed light on the physiological roles of nitric oxide signaling mediated by nonheme iron proteins.

Artificial Metalloproteins with Dinuclear Iron-Hydroxido Centers.

Dinuclear iron centers with a bridging hydroxido or oxido ligand form active sites within a variety of metalloproteins. A key feature of these sites is the ability of the protein to control the structures around the Fe centers, which leads to entatic states that are essential for function. To simulate this controlled environment, artificial proteins have been engineered using biotin-streptavidin (Sav) technology in which Fe complexes from adjacent subunits can assemble to form [FeIII-(µ-OH)-FeIII] cores. The assembly process is promoted by the site-specific localization of the Fe complexes within a subunit through the designed mutation of a tyrosinate side chain to coordinate the Fe centers. An important outcome is that the Sav host can regulate the Fe···Fe separation, which is known to be important for function in natural metalloproteins. Spectroscopic and structural studies from X-ray diffraction methods revealed uncommonly long Fe···Fe separations that change by less than 0.3 Å upon the binding of additional bridging ligands. The structural constraints imposed by the protein host on the di-Fe cores are unique and create examples of active sites having entatic states within engineered artificial metalloproteins.

Omega-3 and its domain-specific effects on cognitive test performance in youths: A meta-analysis.

Omega-3 fatty acids are vital for brain development. The aim of this meta-analysis was to broaden current knowledge of the effects of omega-3 supplementation on cognitive test performance in youths. Randomized controlled trials (RCTs) meeting selection criteria were identified through two independent literature searches on PubMed, Cochrane Library, PsycARTICLES and PsycINFO (last search June 2019). Twenty-nine out of 1126 studies assessing 4247 participants met all selection criteria. A meta-analysis using random-effects model was performed for eight different cognitive domains. This first analysis revealed no main effect of omega-3 fatty acid supplementation on domain-specific cognitive test performance in youths. Subgroup analyses identified beneficial effects of eicosapentaenoic acid (EPA)-rich but not docosahexaenoic acid (DHA)-rich formulations in the domains of long-term memory, working memory and problem solving and a tendency towards beneficial effects in clinical rather than non-clinical populations. Future research should investigate differential effects of EPA and DHA and consider their baseline levels, other nutritional components and interactions with gene variations as potential predictors of response.