Photoacoustic Calorimetry Studies of O-Sensing FixL and (R200, I209) Variants from Reveal Conformational Changes Coupled to Ligand Photodissociation from the Heme-PAS Domain.

FixL is an oxygen-sensing heme-PAS protein that regulates nitrogen fixation in the root nodules of plants. In this paper, we present the first photothermal studies of the full-length wild-type FixL protein from and the first thermodynamic profile of a full-length heme-PAS protein. Photoacoustic calorimetry studies reveal a quadriphasic relaxation for FixL*WT and the five variant proteins (FixL*R200H, FixL*R200Q, FixL*R200E, FixL*R200A, and FixL*I209M) with four intermediates from <20 ns to ∼1.

Engineering to produce -muconic acid from biomass aromatics.

The platform chemical muconic acid (MA) provides facile access to a number of monomers used in the synthesis of commercial plastics. It is also a metabolic intermediate in the β-ketoadipic acid pathway of many bacteria and, therefore, a current target for microbial production from abundant renewable resources via metabolic engineering. This study investigates DSM12444 as a chassis for the production of MA from biomass aromatics.

Recent advances in tuning redox properties of electron transfer centers in metalloenzymes catalyzing oxygen reduction reaction and H oxidation important for fuel cells design.

Current fuel-cell catalysts for oxygen reduction reaction (ORR) and H oxidation use precious metals and, for ORR, require high overpotentials. In contrast, metalloenzymes perform their respective reaction at low overpotentials using earth-abundant metals, making metalloenzymes ideal candidates for inspiring electrocatalytic design. Critical to the success of these enzymes are redox-active metal centers surrounding the enzyme active sites that ensure fast electron transfer (ET) to or away from the active site, by tuning the catalytic potential of the reaction as observed in multicopper oxidases but also in dictating the catalytic bias of the reaction as realized in hydrogenases.

Response of neuroglia to hypoxia-induced oxidative stress using enzymatically crosslinked hydrogels.

Three-dimensional cultures have exciting potential to mimic aspects of healthy and diseased brain tissue to examine the role of physiological conditions on neural biomarkers, as well as disease onset and progression. Hypoxia is associated with oxidative stress, mitochondrial damage, and inflammation, key processes potentially involved in Alzheimer's and multiple sclerosis. We describe the use of an enzymatically-crosslinkable gelatin hydrogel system within a microfluidic device to explore the effects of hypoxia-induced oxidative stress on rat neuroglia, human astrocyte reactivity, and myelin production.

The Heme-Lys Cross-Link in Cytochrome P460 Promotes Catalysis by Enforcing Secondary Coordination Sphere Architecture.

Cytochrome (cyt) P460 is a -type monoheme enzyme found in ammonia-oxidizing bacteria (AOB) and methanotrophs; additionally, genes encoding it have been found in some pathogenic bacteria. Cyt P460 is defined by a unique post-translational modification to the heme macrocycle, where a lysine (Lys) residue covalently attaches to the 13' carbon of the porphyrin, modifying this heme macrocycle into the enzyme's eponymous P460 cofactor, similar to the cofactor found in the enzyme hydroxylamine oxidoreductase. This cross-link imbues the protein with unique spectroscopic properties, the most obvious of which is the enzyme's green color in solution.

Activation of Dioxygen by a Mononuclear Nonheme Iron Complex: Sequential Peroxo, Oxo, and Hydroxo Intermediates.

The activation of dioxygen by Fe(MeTACN)(SSiMe) () is reported. Reaction of with O at -135 °C in 2-MeTHF generates a thiolate-ligated (peroxo)diiron complex Fe(O)(MeTACN)(SSiMe) () that was characterized by UV-vis (λ = 300, 390, 530, 723 nm), Mössbauer (δ = 0.53, |Δ| = 0.

Controlling a burn: outer-sphere gating of hydroxylamine oxidation by a distal base in cytochrome P460.

Ammonia oxidizing bacteria (AOB) use the cytotoxic, energetic molecule hydroxylamine (NHOH) as a source of reducing equivalents for cellular respiration. Despite disproportionation or violent decomposition being typical outcomes of reactions of NHOH with iron, AOB and anammox heme P460 proteins including cytochrome (cyt) P460 and hydroxylamine oxidoreductase (HAO) effect controlled, stepwise oxidation of NHOH to nitric oxide (NO). Curiously, a recently characterized cyt P460 variant from the AOB sp.

A Mononuclear, Nonheme Fe-Piloty's Acid (PhSONHOH) Adduct: An Intermediate in the Production of {FeNO} Complexes from Piloty's Acid.

Reaction of the mononuclear nonheme complex [Fe(CHCN)(N3PyS)]BF (1) with an HNO donor, Piloty's acid (PhSONHOH, P.A.), at low temperature affords a high-spin ( S = 2) Fe-P.

A Nonheme Thiolate-Ligated Cobalt Superoxo Complex: Synthesis and Spectroscopic Characterization, Computational Studies, and Hydrogen Atom Abstraction Reactivity.

The synthesis and characterization of a Co(II) dithiolato complex Co(MeTACN)(SSiMe) (1) are reported. Reaction of 1 with O generates a rare thiolate-ligated cobalt-superoxo species Co(O)(MeTACN)(SSiMe) (2) that was characterized spectroscopically and structurally by resonance Raman, EPR, and X-ray absorption spectroscopies as well as density functional theory. Metal-superoxo species are proposed to S-oxygenate metal-bound thiolate donors in nonheme thiol dioxygenases, but 2 does not lead to S-oxygenation of the intramolecular thiolate donors and does not react with exogenous sulfur donors.

A Nonheme Sulfur-Ligated {FeNO} Complex and Comparison with Redox-Interconvertible {FeNO} and {FeNO} Analogues.

A nonheme {FeNO} complex, [Fe(NO)(N3PyS)] , was synthesized by reversible, one-electron oxidation of an {FeNO} analogue. This complex completes the first known series of sulfur-ligated {FeNO} complexes. All three {FeNO} complexes are readily interconverted by one-electron oxidation/reduction.

Reversible Ligand-Centered Reduction in Low-Coordinate Iron Formazanate Complexes.

Coordination of redox-active ligands to metals is a compelling strategy for making reduced complexes more accessible. In this work, we explore the use of redox-active formazanate ligands in low-coordinate iron chemistry. Reduction of an iron(II) precursor occurs at milder potentials than analogous non-redox-active β-diketiminate complexes, and the reduced three-coordinate formazanate-iron compound is characterized in detail.

Influences of the heme-lysine crosslink in cytochrome P460 over redox catalysis and nitric oxide sensitivity.

Ammonia (NH)-oxidizing bacteria (AOB) derive total energy for life from the multi-electron oxidation of NH to nitrite (NO). One obligate intermediate of this metabolism is hydroxylamine (NHOH), which can be oxidized to the potent greenhouse agent nitrous oxide (NO) by the AOB enzyme cytochrome (cyt) P460. We have now spectroscopically characterized a 6-coordinate (6c) {FeNO} intermediate on the NHOH oxidation pathway of cyt P460.

Nitrosomonas europaea cytochrome P460 is a direct link between nitrification and nitrous oxide emission.

Ammonia oxidizing bacteria (AOB) are major contributors to the emission of nitrous oxide (NO). It has been proposed that NO is produced by reduction of NO. Here, we report that the enzyme cytochrome (cyt) P460 from the AOB Nitrosomonas europaea converts hydroxylamine (NHOH) quantitatively to NO under anaerobic conditions.