Oxidase Chemistry Research Articles

Oxidase‐Type C−H/C−H Coupling Using an Isoquinoline‐Derived Organic Photocatalyst

Oxidase-type oxidation is an attractive strategy in organic synthesis due to the use of O2 as the terminal oxidant. Organic photocatalysis can effect metal-free oxidase chemistry. Nevertheless, current methods are limited in reaction scope, possibly due to the lack of suitable photocatalysts. Here we report an isoquinoline-derived diaryl ketone-type photocatalyst, which has much enhanced absorption of blue and visible light compared to conventional diaryl ketones. This photocatalyst enables dehydrogenative cross-coupling of heteroarenes with unactivated and activated alkanes as well as aldehydes using air as the oxidant. A wide range of heterocycles with various functional groups are suitable substrates. Transient absorption and excited-state quenching experiments point to an unconventional mechanism that involves an excited state “self-quenching” process to generate the N-radical cation form of the sensitizer, which subsequently abstracts a hydrogen atom from the alkane substrate to yield a reactive alkyl radical.

Oxidase-Type C-H/C-H Coupling Using an Isoquinoline-Derived Organic Photocatalyst.

Oxidase‐type oxidation is an attractive strategy in organic synthesis due to the use of O2 as the terminal oxidant. Organic photocatalysis can effect metal‐free oxidase chemistry. Nevertheless, current methods are limited in reaction scope, possibly due to the lack of suitable photocatalysts. Here we report an isoquinoline‐derived diaryl ketone‐type photocatalyst, which has much enhanced absorption of blue and visible light compared to conventional diaryl ketones. This photocatalyst enables dehydrogenative cross‐coupling of heteroarenes with unactivated and activated alkanes as well as aldehydes using air as the oxidant. A wide range of heterocycles with various functional groups are suitable substrates. Transient absorption and excited‐state quenching experiments point to an unconventional mechanism that involves an excited state “self‐quenching” process to generate the N‐radical cation form of the sensitizer, which subsequently abstracts a hydrogen atom from the alkane substrate to yield a reactive alkyl radical.

Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O2 activation

The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to Cα-formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)-FGE is functional in O2 activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor. We establish that the copper atom is coordinated by two active-site cysteine residues in a nearly linear geometry, supporting and extending prior biochemical and structural data. The active cuprous FGE complex was interrogated directly by X-ray absorption spectroscopy. These data unambiguously establish the configuration of the resting enzyme metal center and, importantly, reveal the formation of a three-coordinate tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calculations. Critically, inner-sphere substrate coordination turns on O2 activation at the copper center. These collective results provide a detailed mechanistic framework for understanding why nature chose this structurally unique monocopper active site to catalyze oxidase chemistry for sulfatase activation.

On the Way to a Trisanionic {Cu3 O2 } Core for Oxidase Catalysis: Evidence of an Asymmetric Trinuclear Precursor Stabilized by Perfluoropinacolate Ligands.

CuI complexes of the form K[(R3 P)Cu(pinF )], in which (pinF )2- is the bidentate, oxygen-donating ligand perfluoropinacolate, were synthesized and characterized. Low-temperature oxygenation of the K[(R3 P)Cu(pinF )(PR3 )] species resulted in a trisanionic bis(μ3 -oxo) trinuclear copper(II,II,III) core characterized by UV/Vis spectroscopy (λmax [nm] = 330, 535, 630), cryospray-ionization mass spectrometry, and X-band electron paramagnetic resonance spectroscopy (derivative resonance at 3300 G, Δms =2 at 1500 G). The kinetic behavior of the trimeric {Cu3 O2 } species was quantified by stopped-flow spectroscopy and the associated electronic structures were investigated by DFT calculations. An asymmetric {Cu3 O2 } species, As TpinF , which bears a structure similar to multicopper oxidases, forms prior to full formation of the symmetric trinuclear core, Sy TpinF . The trimer catalytically oxidizes para-hydroquinone to benzoquinone (a form of oxidase chemistry).

Synthetic heme/copper assemblies: toward an understanding of cytochrome c oxidase interactions with dioxygen and nitrogen oxides.

Our long-time niche in synthetic biological inorganic chemistry has been to design ligands and generate coordination complexes of copper or iron ions or both, those reacting with dioxygen (O2) or nitrogen oxides (e.g., nitric oxide (NO(g)) and nitrite (NO2(-))) or both. As inspiration for this work, we turn to mitochondrial cytochrome c oxidase, which is responsible for dioxygen consumption and is also the predominant target for NO(g) and nitrite within mitochondria. In this Account, we highlight recent advances in studying synthetic heme/Cu complexes in two respects. First, there is the design, synthesis, and characterization of new O2 adducts whose further study will add insights into O2 reductive cleavage chemistry. Second, we describe how related heme/Cu constructs reduce nitrite ion to NO(g) or the reverse, oxidize NO(g) to nitrite. The reactions of nitrogen oxides occur as part of CcO's function, which is intimately tied to cellular O2 balance. We had first discovered that reduced heme/Cu compounds react with O2 giving μ-oxo heme-Fe(III)-O-Cu(II)(L) products; their properties are discussed. The O-atom is derived from dioxygen, and interrogations of these systems led to the construction and characterization of three distinctive classes of heme-peroxo complexes, two high-spin and one low-spin species. Recent investigations include a new approach to the synthesis of low-spin heme-peroxo-Cu complexes, employing a "naked" synthon, where the copper ligand denticity and geometric types can be varied. The result is a collection of such complexes; spectroscopic and structural features (by DFT calculations) are described. Some of these compounds are reactive toward reductants/protons effecting subsequent O-O cleavage. This points to how subtle improvements in ligand environment lead to a desired local structure and resulting optimized reactivity, as known to occur at enzyme active sites. The other sector of research is focused on heme/Cu assemblies mediating the redox interplay between nitrite and NO(g). In the nitrite reductase chemistry, the cupric center serves as a Lewis acid, while the heme is the redox active center providing the electron. The orientation of nitrite in approaching the ferrous heme center and N-atom binding are important. Also, detailed spectroscopic and kinetic studies of the NO(g) oxidase chemistry, in excellent agreement with theoretical calculations, reveal the intermediates and key mechanistic steps. Thus, we suggest that both chemical and biochemical heme/Cu-mediated nitrite reductase and NO(g) oxidase chemistry require N-atom binding to a ferrous heme along with cupric ion O-atom coordination, proceeding via a three-membered O-Fe-N chelate ring transition state. These important mechanistic features of heme/Cu systems interconverting NO(g) and nitrite are discussed for the first time.

Performance Analysis of the OneTouch® UltraVue™ Blood Glucose Monitoring System

OneTouch UltraVue is a new meter for self-monitoring of blood glucose that includes a color display, used-strip ejector, and no-button interface. The system uses an electrochemical biosensor technology based on glucose oxidase chemistry to detect glucose concentrations from 20 to 600 mg/dl (1.1 to 33.3 mmol/liter). Accuracy and reproducibility were evaluated over a wide range of glucose concentrations according to standard criteria. Clinical accuracy was assessed by health care providers (HCPs) in two studies and by diabetes patients in the second study. Reference glucose levels were determined by a YSI 2300 analyzer. Same-day reproducibility and day-to-day reproducibility were also evaluated. In the accuracy studies, 99.7% and 98.7% of tests by HCPs and 97.0% of tests by patients were within +/-15 mg/dl (+/-0.8 mmol/liter) of the YSI reference for blood glucose <75 mg/dl (<4.2 mmol/liter), and within +/-20% for blood glucose > or =75 mg/dl (> or =4.2 mmol/liter), respectively. Consensus error grid analysis showed that 99.7% and 95.3% of tests by HCPs and 97.0% of tests by patients fell within zone A (i.e., has no effect on clinical action); all other results were in zone B (i.e., altered clinical action, little or no effect on clinical outcome). In the reproducibility studies, the standard deviation was <1.5 mg/dl (<0.1 mmol/liter) for glucose concentrations <100 mg/dl (<5.6 mmol/liter), and the coefficient of variation was <2% for concentrations > or = 100 mg/dl (> or =5.6 mmol/liter). OneTouch UltraVue meets standard acceptability criteria for accuracy and reproducibility across a wide range of glucose concentrations. Its simple interface and lack of contact with used strips make it a viable option for older patients and their caregivers.

A single mutation in the castor Delta9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry.

Sequence analysis of the diiron cluster-containing soluble desaturases suggests they are unrelated to other diiron enzymes; however, structural alignment of the core four-helix bundle of desaturases to other diiron enzymes reveals a conserved iron binding motif with similar spacing in all enzymes of this structural class, implying a common evolutionary ancestry. Detailed structural comparison of the castor desaturase with that of a peroxidase, rubrerythrin, shows remarkable conservation of both identity and geometry of residues surrounding the diiron center, with the exception of residue 199. Position 199 is occupied by a threonine in the castor desaturase, but the equivalent position in rubrerythrin contains a glutamic acid. We previously hypothesized that a carboxylate in this location facilitates oxidase chemistry in rubrerythrin by the close apposition of a residue capable of facilitating proton transfer to the activated oxygen (in a hydrophobic cavity adjacent to the diiron center based on the crystal structure of the oxygen-binding mimic azide). Here we report that desaturase mutant T199D binds substrate but its desaturase activity decreases by approximately 2 x 10(3)-fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect to WT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-A crystal structure of T199D reveals active-site geometry remarkably similar to that of rubrerythrin, consistent with its enhanced function as an oxidase enzyme. That a single amino acid substitution can switch reactivity from desaturation to oxidation provides experimental support for the hypothesis that the desaturase evolved from an ancestral oxidase enzyme.

A Hospital System Glucose Meter That Produces Plasma-Equivalent Values from Capillary, Venous, and Arterial Blood

The use of point-of-care meters for bedside blood glucose monitoring has become common in hospitalized patients. The management of patients in critical care units, those receiving aggressive supportive therapy, and those with type 1 or 2 diabetes may require frequent testing of their blood glucose concentrations (1)(2)(3). Bedside monitoring is often done with capillary whole blood from a fingerstick, and these whole-blood glucose meters produce values 8–10% lower than those in plasma (4). However, most clinical laboratory instruments measure glucose concentrations in plasma or serum, not whole blood. A whole-blood glucose meter that reports a plasma-equivalent result might be useful to reduce possible confusion when comparing glucose meter and laboratory glucose values. Here, we evaluated the SureStep®-Pro Hospital System, a reflectance meter that uses glucose oxidase chemistry to report plasma-equivalent glucose concentrations from capillary, venous, and arterial whole blood. We compared whole-blood and plasma glucose concentrations measured by the SureStep-Pro Hospital System (LifeScan) with values from the One Touch® II Hospital System (LifeScan), the Yellow Springs Instrument Model 2700 Select Biochemistry Analyzer (YSI), and the Vitros 750 Analyzer (Johnson & Johnson). Plasma equivalent results on the SureStep-Pro Hospital …

The roles of active site hydrogen bonding in cytochrome P-450cam as revealed by site-directed mutagenesis.

The role of the active site hydrogen bond of cytochrome P-450cam has been studied utilizing a combination of site-directed mutagenesis and substrate analogues with altered hydrogen bonding capabilities. Cytochrome P-450cam normally catalyzes the regiospecific hydroxylation of the monoterpene camphor. The x-ray crystal structure of this soluble bacterial cytochrome P-450 (Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and Kraut, J. (1985) J. Biol. Chem. 260, 16122-16128) indicates a specific hydrogen bond between tyrosine 96 and the carbonyl moiety of the camphor substrate. The site-directed mutant in which tyrosine 96 has been changed to a phenylalanine and the substrate analogues thiocamphor and camphane have been used to probe this interaction in several aspects of catalysis. At room temperature, both the mutant enzyme with camphor and the wild type enzyme with thiocamphor bound result in 59 and 65% high-spin ferric enzyme as compared to the 95% high spin population obtained with native enzyme and camphor as substrate. The equilibrium dissociation constant is moderately increased, from 1.6 microM for the wild type protein to 3.0 and 3.3 microM for wild type-thiocamphor and mutant-camphor complexes, respectively. Camphane bound to cytochrome P-450cam exhibits a larger decrease in high spin fraction (45%) and a correspondingly larger KD (46 microM), suggesting that the carbonyl moiety of camphor plays an important steric role in addition to its interaction as a hydrogen bond acceptor. The absolute regioselectivity of the mutant enzyme, and of the wild type enzyme with thiocamphor, is lost resulting in production of several hydroxylated products in addition to the 5-exo-hydroxy isomer. Based on rates of NADH oxidation, comparison of the substrate specificity for these systems (kcat/KD) indicates a 5- and 7-fold decrease in specificity for the mutant enzyme and thiocamphor-wild type complex, respectively. The replacement of the cytochrome P-450cam active site tyrosine with phenylalanine does not affect the branching ratio of monooxygenase versus oxidase chemistry or peroxygenase activity (Atkins, W.M., and Sligar, S.G. (1987) J. Am. Chem. Soc. 109, 3754-3760).