Stopped-flow Spectrophotometry Research Articles

A flexible linker of 8-amino acids between the membrane binding segment and the FMN domain of cytochrome P450 reductase is necessary for optimal activity

The diflavin NADPH-cytochrome P450 reductase (CYPOR) plays a critical role in human cytochrome P450 (CYP) activity by sequentially delivering two electrons from NADPH to CYP enzymes during catalysis. Although electron transfer to forty-eight human CYP enzymes by the FMN hydroquinone of CYPOR is well-known, the role of the linker between the NH2-terminus membrane-binding domain (MBD) and FMN domain in supporting the activity of P450 enzymes remains poorly understood. Here we demonstrate that a linker with at least eight residues is required to form a functional CYPOR-CYP2B4 complex. The linker has been shortened in two amino-acid increments from Phe44 to Ile57 using site directed mutagenesis. The ability of the deletion mutants to support cytochrome P450 2B4 (CYP2B4) catalysis and reduce ferric CYP2B4 was determined using an in vitro assay and stopped-flow spectrophotometry. Steady-state enzyme kinetics showed that shortening the linker by 8–14 amino acids inhibited (63–99%) the ability of CYPOR to support CYP2B4 activity and significantly increased the Km of CYPOR for CYP2B4. In addition, the reductase mutants decreased the rate of reduction of ferric CYP2B4 (46–95%) compared to wildtype when the linker was shortened by 8–14 residues. These results indicate that a linker with a minimum length of eight residues is necessary to enable the FMN domain of reductase to interact with CYP2B4 to form a catalytically competent complex. Our study provides evidence that the length of the MBD-FMN domain linker is a major determinant of the ability of CYPOR to support CYP catalysis and drug metabolism by P450 enzymes. PreambleThis manuscript is dedicated in memory of Dr. James R. Kincaid who was the doctoral advisor to Dr. Freeborn Rwere and a longtime collaborator and friend of Dr. Lucy Waskell. Dr. James R. Kincaid was a distinguished professor of chemistry specializing in resonance Raman (rR) studies of heme proteins. He inspired Dr. Rwere (a Zimbabwean native) and three other Zimbabweans (Dr. Remigio Usai, Dr. Daniel Kaluka and Ms. Munyaradzi E. Manyumwa) to use lasers to document subtle changes occurring at heme active site of globin proteins (myoglobin and hemoglobin) and cytochrome P450 enzymes. Dr. Rwere appreciate his contributions to the development of talented Black scientists from Africa.

On the Mechanisms of Hypohalous Acid Formation and Electrophilic Halogenation by Non-Native Halogenases.

Enzymatic electrophilic halogenation is a mild tool for functionalization of diverse organic compounds. Only a few groups of native halogenases are capable of catalyzing such a reaction. In this study, we used a mechanism-guided strategy to discover the electrophilic halogenation activity catalyzed by non-native halogenases. As the ability to form a hypohalous acid (HOX) is key for halogenation, flavin-dependent monooxygenases/oxidases capable of forming C4a-hydroperoxyflavin (FlC4a-OOH), such as dehalogenase, hydroxylases, luciferase and pyranose-2-oxidase (P2O), and flavin reductase capable of forming H2O2 were explored for their abilities to generate HOX in situ. Transient kinetic analyses using stopped-flow spectrophotometry/fluorometry and product analysis indicate that FlC4a-OOH in dehalogenases, selected hydroxylases and luciferases, but not in P2O can form HOX; however, the HOX generated from FlC4a-OOH cannot halogenate their substrates. Remarkably, in situ H2O2 generated by P2O can form HOI and also iodinate various compounds. Because not all enzymes capable of forming FlC4a-OOH can react with halides to form HOX, QM/MM calculations, site-directed mutagenesis and structural analysis were carried out to elucidate the mechanism underlying HOX formation and characterize the active site environment. Our findings shed light on identifying new halogenase scaffolds besides the currently known enzymes and have invoked a new mode of chemoenzymatic halogenation.

Peroxidasin Inhibition by Phloroglucinol and Other Peroxidase Inhibitors.

Human peroxidasin (PXDN) is a ubiquitous peroxidase enzyme expressed in most tissues in the body. PXDN represents an interesting therapeutic target for inhibition, as it plays a role in numerous pathologies, including cardiovascular disease, cancer and fibrosis. Like other peroxidases, PXDN generates hypohalous acids and free radical species, thereby facilitating oxidative modifications of numerous biomolecules. We have studied the inhibition of PXDN halogenation and peroxidase activity by phloroglucinol and 14 other peroxidase inhibitors. Although a number of compounds on their own potently inhibited PXDN halogenation activity, only five were effective in the presence of a peroxidase substrate with IC50 values in the low μM range. Using sequential stopped-flow spectrophotometry, we examined the mechanisms of inhibition for several compounds. Phloroglucinol was the most potent inhibitor with a nanomolar IC50 for purified PXDN and IC50 values of 0.95 μM and 1.6 μM for the inhibition of hypobromous acid (HOBr)-mediated collagen IV cross-linking in a decellularized extracellular matrix and a cell culture model. Other compounds were less effective in these models. Most interestingly, phloroglucinol was identified to irreversibly inhibit PXDN, either by mechanism-based inhibition or tight binding. Our work has highlighted phloroglucinol as a promising lead compound for the design of highly specific PXDN inhibitors and the assays used in this study provide a suitable approach for high-throughput screening of PXDN inhibitors.

Amino Acid Residues Controlling Domain Interaction and Interdomain Electron Transfer in Cellobiose Dehydrogenase.

The function of cellobiose dehydrogenase (CDH) in biosensors, biofuel cells, and as a physiological redox partner of lytic polysaccharide monooxygenase (LPMO) is based on its role as an electron donor. Before donating electrons to LPMO or electrodes, an interdomain electron transfer from the catalytic FAD-containing dehydrogenase domain to the electron shuttling cytochrome domain of CDH is required. This study investigates the role of two crucial amino acids located at the dehydrogenase domain on domain interaction and interdomain electron transfer by structure-based engineering. The electron transfer kinetics of wild-type Myriococcum thermophilum CDH and its variants M309A, R698S, and M309A/R698S were analyzed by stopped-flow spectrophotometry and structural effects were studied by small-angle X-ray scattering. The data show that R698 is essential to pull the cytochrome domain close to the dehydrogenase domain and orient the heme propionate group towards the FAD, while M309 is an integral part of the electron transfer pathway - its mutation reducing the interdomain electron transfer 10-fold. Structural models and molecular dynamics simulations pinpoint the action of these two residues on the domain interaction and interdomain electron transfer.

Reactivity and mechanism of the reactions of 4-methylbenzoquinone with amino acid residues in β-lactoglobulin: A kinetic and product investigation

Quinones, produced by the oxidation of phenolic compounds, covalently bind to nucleophilic groups on amino acids or proteins. In this study, the reactions of 4-methylbenzoquinone (4MBQ) with β-lactoglobulin (β-LG) and amino acids at neutral pH were investigated. LC-MS analysis revealed that Cys121 was likely the most modified residue in β-LG. Identification of reaction products by LC-MS/MS showed that Michael addition occurred in all reactions with amino acids tested. The formation of Schiff base and a di-adduct was found in His and Trp samples. Apparent second-order rate constants (k2) were determined at 25 °C and pH 7.0 by stopped-flow spectrophotometry. The rate of reactions decreased in the order: β-LG > His > Trp > Arg > Nα-acetyl His > Nα-acetyl Arg > Nα-acetyl Trp. The rate constants correlated with the pKa values of the amino acids, showing that the amount of unprotonated amine is the major factor determining the reactivity.

The Role of the si-Face Tyrosine of a Homodimeric Ferredoxin-NADP+ Oxidoreductase from Bacillus subtilis during Complex Formation and Redox Equivalent Transfer with NADP+/H and Ferredoxin.

In the crystal structure of ferredoxin-NADP+ oxidoreductase from Bacillus subtilis (BsFNR), Tyr50 stacks on the si-face of the isoalloxazine ring portion of the FAD prosthetic group. This configuration is highly conserved among the homodimeric ferredoxin-NAD(P)+ oxidoreductases (FNR) from Gram-positive bacteria and photosynthetic bacteria. In this report, pre-steady state reactions of Tyr50 variants with NADP+/NADPH and ferredoxin from B. subtilis (BsFd) were examined with stopped-flow spectrophotometry to assess the effects of the mutation on the formation of FNR-substrate complexes and following redox equivalent transfer. Mixing oxidized BsFNRs with NADPH resulted in a rapid complex formation followed by a rate-limiting hydride transfer. The substitution substantially modulated the intensity of the charge transfer absorption band and decreased the observed hydride transfer rates compared to the wild type. Reduction of the Y50W mutant by NADPH proceeded in a monophasic manner, while the Y50G and Y50S mutants did in biphasic phases. The reduced Tyr50 mutants hardly promoted the reduction of NADP+. Mixing oxidized BsFNRs with reduced BsFd resulted in the reduction of the FNRs. The observed FNR reduction rates of the three variants were comparable, but in the Y50G and Y50S mutants, the amount of the reduced FNR at the rapid phase was decreased, and a slow FNR reduction phase was observed. The obtained results suggest that the replacements of Tyr50 with Gly and Ser permitted the conformational change in the reduced form, which induced an asymmetric kinetic behavior between the protomers of the homodimeric BsFNR.

Model-Driven Design of Redox Mediators: Quantifying the Impact of Quinone Structure on Bioelectrocatalytic Activity with Glucose Oxidase.

Successful application of emerging bioelectrocatalysis technologies depends upon an efficient electrochemical interaction between redox enzymes as biocatalysts and conductive electrode surfaces. One approach to establishing such enzyme-electrode interfaces utilizes small redox-active molecules to act as electron mediators between an enzyme-active site and the electrode surface. While redox mediators have been successfully used in bioelectrocatalysis applications ranging from enzymatic electrosynthesis to enzymatic biofuel cells, they are often selected using a guess-and-check approach. Herein, we identify structure-function relationships in redox mediators that describe the bimolecular rate constant for its reaction with a model enzyme, glucose oxidase (GOx). Based on a library of quinone-based redox mediators, a quantitative structure-activity relationship (QSAR) model is developed to describe the importance of mediator redox potential and projected molecular area as two key parameters for predicting the activity of quinone/GOx-based electroenzymatic systems. Additionally, rapid scan stopped-flow spectrophotometry was used to provide fundamental insights into the kinetics and the stoichiometry of reactions between different quinones and the flavin adenine dinucleotide (FAD+/FADH2) cofactor of GOx. This work provides a critical foundation for both designing new enzyme-electrode interfaces and understanding the role that quinone structure plays in altering electron flux in electroenzymatic reactions.

The interfacial “push and pull” competitive complexation and enhanced separation of praseodymium and neodymium

The separation of praseodymium (Pr) and neodymium (Nd) is particularly challenging within the lanthanide series. A new strategy was developed for enhanced separation of Pr and Nd, using interfacial “push and pull” complexation induced by a kinetic effect. We found that the specific addition order of diethylenetriaminepentaacetic acid (DTPA) had an obvious influence on the separation behaviors of Pr/Nd. Notably, the separation coefficient was up to 3.25 with the addition of DTPA when the extraction process just started. This suggested that different complexation rates decided differences of extraction kinetics between Pr and Nd. It revealed differences in complexation rates of adjacent RE(III) with DTPA and dissociation rates of RE-DTPA complexes by stopped-flow spectrophotometry. The results indicated that the complexation rate constant of Nd3+ was higher than Pr3+. On contrary, the dissociation rate constant of Nd-DTPA was smaller than Pr-DTPA. Molecular dynamics (MD) simulations showed that the interfacial “push and pull” complexation effect led to differences in interfacial molecule arrangements and diffusion mass transfer rates near the interface. And thus, the separation of Pr/Nd can be enhanced. This research provides an efficient strategy for the purification of metals that are difficult to separate.

The Kinetics of the Redox Reaction of Platinum(IV) Ions with Ascorbic Acid in the Presence of Oxygen

In this work, the kinetics of the redox reaction between platinum(IV) chloride complex ions and ascorbic acid is studied. The reduction process of Pt(IV) to Pt(II) ions was carried out at different reagent concentrations and environmental conditions, i.e., pH (2.2–5.1), temperature (20–40 °C), ionic strength (I = 0.00–0.40 M) and concentrations of chloride ions (0.00–0.40 M). The kinetic traces during the reduction process were registered using stopped-flow spectrophotometry. Based on the kinetic traces, the rate constants were determined, and the kinetic equations were proposed. It was shown that in the mild acidic medium (pH = 2.5), the reduction process of Pt(IV) to Pt(II) ions is more complex in the presence of oxygen dissolved in the aqueous solutions. For these processes, the values of the enthalpy and entropy of activation were determined. Moreover, the mechanism of the reduction of Pt(IV) to Pt(II) ions was proposed. The presented results give an overview of the process of the synthesis of platinum nanoparticles in the solution containing oxygen, in which the reduction process of Pt(IV) to Pt(II) ions is the first step.

Mechanism of the Multistep Catalytic Cycle of 6-Hydroxynicotinate 3-Monooxygenase Revealed by Global Kinetic Analysis

The class A flavoenzyme 6-hydroxynicotinate 3-monooxygenase (NicC) catalyzes a rare decarboxylative hydroxylation reaction in the degradation of nicotinate by aerobic bacteria. While the structure and critical residues involved in catalysis have been reported, the mechanism of this multistep enzyme has yet to be determined. A kinetic understanding of the NicC mechanism would enable comparison to other phenolic hydroxylases and illuminate its bioengineering potential for remediation of N-heterocyclic aromatic compounds. Toward these goals, transient state kinetic analyses by stopped-flow spectrophotometry were utilized to follow rapid changes in flavoenzyme absorbance spectra during all three stages of NicC catalysis: (1) 6-HNA binding; (2) NADH binding and FAD reduction; and (3) O2 binding with C4a-adduct formation, substrate hydroxylation, and FAD regeneration. Global kinetic simulations by numeric integration were used to supplement analytical fitting of time-resolved data and establish a kinetic mechanism. Results indicate that 6-HNA binding is a two-step process that substantially increases the affinity of NicC for NADH and enables the formation of a charge-transfer-complex intermediate to enhance the rate of flavin reduction. Singular value decomposition of the time-resolved spectra during the reaction of the substrate-bound, reduced enzyme with dioxygen provides evidence for the involvement of C4a-hydroperoxy-flavin and C4a-hydroxy-flavin intermediates in NicC catalysis. Global analysis of the full kinetic mechanism suggests that steady-state catalytic turnover is partially limited by substrate hydroxylation and C4a-hydroxy-flavin dehydration to regenerate the flavoenzyme. Insights gleaned from the kinetic model and determined microscopic rate constants provide a fundamental basis for understanding NicC's substrate specificity and reactivity.

Manganese(II) complexes of 1,1′-bis[(pyridin-2-yl)methyl)]-2,2′-bipiperidine (PYBP): Synthesis, structure, catalytic properties in alkene epoxidation with hydrogen peroxide, and related mechanistic studies

Several manganese(II) complexes with the stereoisomers of ligand PYBP (1,1′-bis[(pyridin-2-yl)methyl]-2,2′-bipiperidine) and different anions were prepared and characterized by X-ray diffractometry. Complex [MnII(rac-PYBP)]2+ (1) was found to be an efficient catalyst of alkene epoxidation by hydrogen peroxide in the presence of acetic acid in acetonitrile at room temperature. Cyclooctene was converted to its epoxide with up to 91 % yield, 99.6 % selectivity, and the turnover number of 180 within 5 min. Fast epoxidations of cyclohexene, 1-decene, styrene, and cis-stilbene were also achieved. Isomeric complex [MnII(meso-PYBP)]2+ (2) was catalytically inactive under the same experimental conditions. Stopped-flow spectrophotometry and freeze-quenched EPR spectra show that complex 2 is not oxidized by H2O2 in the presence of acetic acid (AcOH) but instead undergoes partial ligand protonation and liberation of the Mn2+ cations due to the relatively poor chelating ability of ligand meso-PYBP. The rac-PYBP isomer acts as a better ligand and retains the coordinated Mn center when complex 1 is treated with the H2O2/AcOH mixture in acetonitrile solution yielding a mixture of intensely colored intermediates likely involving MnIII, MnIV, and MnV complexes. Magnetic susceptibility measurements, UV–vis and EPR spectra suggest that dinuclear complexes [MnIII2(μ-O)(μ-OAc)(rac-PYBP)2]3+ and [MnIIIMnIV(μ-O)2(rac-PYBP)2]3+ gradually accumulate in the reaction mixture as inactivated states of the catalyst. Complex 1 also causes fast decomposition of hydrogen peroxide into O2 gas and H2O, which competes with the epoxidation of alkenes and requires gradual addition of H2O2 for its efficient use. The catalytic activity of complex 1 is strongly influenced by its counterions and decreases in order ClO4− ≈ SbF6− > NO3– > Cl− indicating that labile ligands in the coordination sphere of Mn are required for the activation of H2O2.

Fast and biphasic 8-nitroguanine production from guanine and peroxynitrite.

Guanine (Gua), among purines, is a preferred oxidation/nitration target because of its low one-electron redox potential. The reactive oxygen/nitrogen species peroxynitrite (ONOO-), produced in vivo by the reaction between nitric oxide (•NO) and superoxide radical (O2•‒), is responsible for several oxidative modifications in biomolecules, including nitration, nitrosation, oxidation, and peroxidation. In particular, the nitration of Gua, although detected, as well as its reaction kinetics have been seldom investigated. Thus, we studied the concentration- and temperature-dependent formation of 8-nitroguanine (8-NitroGua) in phosphate buffer (pH 7.40) using stopped-flow spectrophotometry. Traces showed a biexponential behavior, with best-fit rate constants: kfast=4.4 s-1 and kslow=0.41 s-1 (30°C, 400μM both Gua and ONOO-). kfast increased linearly with the concentration of both reactants whereas kslow was concentration-independent. Linear regression analysis of kfast as a function of Gua and ONOO- concentration yielded values of 2.5-6.3×103M-1s-1 and 1.5-3.5 s-1 for the second-order (slope) and first-order (ordinate) rate constants, respectively (30°C). Since ONOO- is a short-lived species, its decay kinetics was also taken into account for this analysis. The 8-NitroGua product was stable for at least 4h, so no spontaneous denitration was observed. Stopped-flow assays using antioxidants and free-radical scavengers suggested a mixed direct/indirect reaction mechanism for 8-NitroGua formation. Gua nitration by ONOO- was also observed in the presence of physiologically relevant CO2 concentrations. The reaction product identity, its yield (∼4.2%, with 400μM ONOO- and 200μM Gua), and the reaction mechanism were unequivocally determined by HPLC-MS/MS experiments. In conclusion, 8-NitroGua production at physiologic pH reached significant levels in a few hundred milliseconds, suggesting that the process might be kinetically relevant in vivo and can likely cause permanent nitrative damage to DNA bases.

Designing Enzymatic Redox Mediators: Quantifying the Impact of Molecular Structure on Biocatalytic Activity

Enzymatic bioelectrocatalysis involves the application of enzymes to facilitate the conversion of chemical to electrical energy. This approach has been widely applied in the fields of biosensing, biofuel cells, and to a lesser degree the synthesis of fine chemicals. While direct electrochemical communication between the enzymes and electrodes is generally preferable, it is often the case that efficient electron transfer rates can only be achieved when redox mediators are employed to shuttle electrons reversibly from the redox site of the enzyme to the electrode interface. Unfortunately, there still exist many oxidoreductases for which no effective exogenous mediator is known. This may be due to insufficient understanding of the role that molecular structure of the mediator plays in determining its ability to facilitate electron transfer to a given enzyme. Consequently, selection and/or design of novel redox mediators remains a challenge that is often accomplished using a “guess and check” approach to maximize electron transfer rates. Developing a rapid, and convenient method of predicting the role played by other structural and chemical features beyond redox potential of a mediator is an important goal in advancing mediated bioelectrocatalysis. This talk will describe our recent efforts in identifying structure - function relationships of redox mediators that control efficient electron transfer rates. I will highlight results from stopped-flow spectrophotometry experiments which review the nature of electron transfer events between quinone redox couples in altering bioelectrocatalytic activity.

Flavin binding affinity and initial kinetic characterization of DnmZ, a flavin‐dependent N‐oxygenase

Doxorubicin is an anthracycline chemotherapeutic produced by Streptomyces peucetius. It is an effective chemotherapeutic but has severe side effects and high toxicity, and the details of its biosynthetic pathway are largely unknown. Baumycin is a derivatized form of doxorubicin with an additional seven‐carbon acetal moiety attached to the anthracycline backbone of the molecule. In this work, we report our progress on the characterization of DnmZ, a Class D flavin monooxygenase involved in formation of the baumycin acetal. DnmZ is a N‐oxygenase that oxidizes a sugar‐linked exocyclic amine, triggering a nonenzymatic retro oxime‐aldol cleavage of the hexose. We aim to characterize the kinetics of the DnmZ‐catalyzed reaction between flavin and oxygen using stopped flow spectrophotometry. Results from this research can aid in the development of derivatized anthracyclines and the understanding of the class of enzymes known as the class D flavin monooxygenases.Our transient‐state experiments confirm that DnmZ catalyzes the formation of the C4a‐(hydro)peroxyflavin intermediate (C4a‐OOH). We have identified the appropriate wavelengths to monitor to observe C4a‐OOH formation (378nm) and elimination (450 and 480 nm). With this information we have quantitated flavin binding and determined that DnmZ’s binding affinity for flavin is approximately 13 mM, which compares well with other Class D monooxygenases. Our preliminary results suggest that increasing pH speeds up C4a‐OOH elimination but has no effect on the intermediate’s formation. We are also investigating the effect of mutating active site residues on C4a‐OOH formation and elimination.

Exploring The Promiscuity Potential Of 6‐Hydroxynicotinate‐3‐Monooxygenase: Consequences To Catalysis Of Adding A Nitrogen At C5 Within The Substrate’s Aromatic Ring

Bacterial degradation of nicotinic acid is a model for exploring the bioremediation potential of soil bacteria. One enzyme within this catabolic pathway is 6‐hydroxynicotinate‐3‐monooxygenase (NicC), a group A flavoenzyme that catalyzes the conversion of 6‐hydroxynicotinate (6HNA) to 2,5‐dihydroxypyridine. This decarboxylative hydroxylation reaction is relatively unique in biology. We recently published a study highlighting NicC’s ability to catalyze the decarboxylation and hydroxylation reaction with alternative substrates including 4‐hydroxybenzoate (4HBA) and 5‐chloro‐6‐hydroxynicotinate (5‐Cl‐6HNA). To further explore the structural basis of a substrate that enables tight binding and catalysis with NicC, 2‐hydroxypyrimidine‐5‐caboxylate (2HM5A) was characterized. This 6‐HNA analogue with an extra N atom at C5 was chosen due to its fundamental symmetry and comparable electron distribution as 5‐Cl‐6HNA. Analyses of NicC with 2HM5A show that the expected product, 2,5‐dihydroxypyrimidine (isouracil), is generated, with H2O2 occurring at a mol fraction of about 0.07, indicating that uncoupling between NADH oxidation and substrate hydroxylation is minimal. Equilibrium titration of NicC with 2HM5A by changes in A‐450 nm measure the Kd = 39 ± 6 µM, indicating that this substrate is bound more tightly than 6‐HNA (Kd= 58 ± 12 µM) but less tightly than 5‐Cl‐6‐HNA (Kd= 7 ± 2 µM). Interestingly, the overall ΔA450 elicited by the binding of 2HM5A (0.018) was smaller in comparison to the ΔA450 observed for the binding of 5‐Cl‐6HNA (0.07) or 6‐HNA (0.05). Attenuated flavin movement due the extra nitrogen of the pyrimidine ring in 2HM5A H‐bonding to the isoalloxazine ring may explain this observation. The kinetics of binding 2HM5A by NicC measured by stopped flow spectrophotometry shows that this analogue binds by a two‐step mechanism, with a nearly ten‐fold higher initial affinity for NicC prior to isomerization (ionization) than 6‐HNA. This added affinity may again be explainable by the added H‐bonding potential of this analogue. In the second step of binding, 2HM5A was observed to have a rate constant similar to 5‐Cl‐6HNA (k2= 220 s‐1), possibly indicating that increased electron density at/near the C5 position accelerates this phase of the binding mechanism. How 2HM5A affects NicC’s mechanism of flavin reduction and oxidation will also be presented. These findings provide further evidence of this monooxygenase’s potential to be engineered to hydroxylate other N‐heterocyclic aromatic compounds in bioremediation.

Determination of the pH dependence, substrate specificity, and turnovers of alternative substrates for human ornithine aminotransferase

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and occurs predominantly in patients with underlying chronic liver diseases. Over the past decade, human ornithine aminotransferase (hOAT), which is an enzyme that catalyzes the metabolic conversion of ornithine into an intermediate for proline or glutamate synthesis, has been found to be overexpressed in HCC cells. hOAT has since emerged as a promising target for novel anticancer therapies, especially for the ongoing rational design effort to discover mechanism-based inactivators (MBIs). Despite the significance of hOAT in human metabolism and its clinical potential as a drug target against HCC, there are significant knowledge deficits with regard to its catalytic mechanism and structural characteristics. Ongoing MBI design efforts require in-depth knowledge of the enzyme active site, in particular, pKa values of potential nucleophiles and residues necessary for the molecular recognition of ligands. Here, we conducted a study detailing the fundamental active-site properties of hOAT using stopped-flow spectrophotometry and X-ray crystallography. Our results quantitatively revealed the pH dependence of the multistep reaction mechanism and illuminated the roles of ornithine α-amino and δ-amino groups in substrate recognition and in facilitating catalytic turnover. These findings provided insights of the catalytic mechanism that could benefit the rational design of MBIs against hOAT. In addition, substrate recognition and turnover of several fragment-sized alternative substrates of hOATs, which could serve as structural templates for MBI design, were also elucidated.

Oxidized liposomal artificial red blood cells rescue azide-poisoned mice from lethal toxidrome by recovering cytochrome c oxidase activity

Exposure to azide compounds causes inhibition of cytochrome c oxidase in the mitochondria, leading to acute lethal poisoning. Although urgent pharmaceutical intervention is required for rescue from azide poisoning, no antidote exists worldwide. We hypothesized that methemoglobin (metHb) can be a promising material as an antidote for azide poisoning because metHb can strongly bind to azide. However, metHb administration is not feasible owing to in vivo instability and toxicity. We aimed to develop a feasible metHb-based antidote for azide poisoning, in which the inner hemoglobin of liposomal artificial red blood cells is oxidized by mixing with sodium nitrite. From the stopped-flow spectrophotometry analysis, as-prepared oxidized liposomal artificial red blood cells, which encapsulated metHb into the liposome (metHb@Lipo), the binding affinity of metHb@Lipo to azide was comparable to that of bare metHb. In addition, detoxification by metHb@Lipo increased the survival rate in lethal azide-poisoned model mice with recovery of cytochrome c oxidase activity, leading to the amelioration of acidosis and tissue oxidation. Furthermore, metHb@Lipo detoxification functioned even after 1 year of storage in a ready-to-use formulation. These results indicate that oxidized liposomal artificial red blood cells are a potent antidote for azide poisoning with favorable properties for use in critical care medicine.

New Insight into the Protein‐based Cofactor of Mycobacterium tuberculosis KatG: Toward Better Understanding an Unusual Catalase Mechanism

Catalase-peroxidases (KatG) has been engineered by nature to exhibit dual functionalism of catalase and peroxidase mechanisms, serving to protect the organisms that carry it against peroxide-dependent oxidative damage. Despite bearing no resemblance to monofunctional (i.e., typical) catalases, KatGs have robust catalase activity due at least in part to a novel covalent linkage between three side chains (by Mycobacterium tuberculosis KatG [MtKatG] numbering, Met-255, Tyr-229, and Trp-107) (MYW). This MYW cofactor redox cycles between its radical (MYW•+) and fully covalent states, enabling KatG to leverage heme intermediates for catalatic O2 production. However, the molecular mechanism by which the adduct is formed and how this unique structure contributes to overall catalytic mechanism of KatG is yet to be explored. Here, optical stopped-flow spectrophotometry, rapid freeze-quench EPR spectroscopy and mutagenesis have been used to investigate the mechanism of MYW adduct formation. We have expressed and purified MtKatG lacking heme and reconstituted with the cofactor after purification. This produces KatG lacking the MYW crosslink, allowing us to monitor its formation upon reaction with peroxides. Under multiple-turnover conditions using H2O2, optical stopped-flow experiments showed an initial appearance of a high-valent ferryl-like (FeIV=O) intermediate instead of the typical FeIII-O2•- -like steady-state intermediate of KatG's catalase activity. Nevertheless, in contrast to catalase-negative canonical heme peroxidases, full catalase H2O2 decomposition did emerge, returning the enzyme to its FeIII state. EPR experiments revealed that an admixture of radical species appeared before MYW cofactor radical intermediate, suggestive of the preferred site of crosslink initiation. In addition to that, KatG variants lacking the MYW adduct (M255I, Y229F and W107F) were also constructed, expressed, purified, and reconstituted with heme. Stopped flow and UV-vis analysis of these variants showed disrupted catalase activities and incomplete catalatic turnover. These observations strongly suggest that, these distal side residues actively participate in intramolecular electron transfer, thereby fulfilling a mechanistic role in KatG catalase mechanism. Our reconstituted KatG proteins may permit investigation of radical transfer reactions leading to formation of KatG's novel MYW cofactor as well as the influence of other protein radical transfer reactions on that process.

A heme•DNAzyme activated by hydrogen peroxide catalytically oxidizes thioethers by direct oxygen atom transfer rather than by a Compound I-like intermediate.

Hemin [Fe(III)-protoporphyrin IX] is known to bind tightly to single-stranded DNA and RNA molecules that fold into G-quadruplexes (GQ). Such complexes are strongly activated for oxidative catalysis. These heme•DNAzymes and ribozymes have found broad utility in bioanalytical and medicinal chemistry and have also been shown to occur within living cells. However, how a GQ is able to activate hemin is poorly understood. Herein, we report fast kinetic measurements (using stopped-flow UV–vis spectrophotometry) to identify the H2O2-generated activated heme species within a heme•DNAzyme that is active for the oxidation of a thioether substrate, dibenzothiophene (DBT). Singular value decomposition and global fitting analysis was used to analyze the kinetic data, with the results being consistent with the heme•DNAzyme's DBT oxidation being catalyzed by the initial Fe(III)heme–H2O2 complex. Such a complex has been predicted computationally to be a powerful oxidant for thioether substrates. In the heme•DNAzyme, the DNA GQ enhances both the kinetics of formation of the active intermediate as well as the oxidation step of DBT by the active intermediate. We show, using both stopped flow spectrophotometry and EPR measurements, that a classic Compound I is not observable during the catalytic cycle for thioether sulfoxidation.

Structural Basis of the Stereochemistry of Inhibition of Tryptophan Synthase by Tryptophan and Derivatives.

We have examined the reaction of Salmonella enterica serovar typhimurium tryptophan (Trp) synthase α2β2 complex with l-Trp, d-Trp, oxindolyl-l-alanine (OIA), and dioxindolyl-l-alanine (DOA) in the presence of disodium (dl)-α-glycerol phosphate (GP), using stopped-flow spectrophotometry and X-ray crystallography. All structures contained the d-isomer of GP bound at the α-active site. (3S)-OIA reacts with the pyridoxal-5'-phosphate (PLP) of Trp synthase to form a mixture of external aldimine and quinonoid complexes. The α-carboxylate of OIA rotates about 90° to become planar with the PLP when the quinonoid complex is formed, resulting in a conformational change in the loop of residues 110-115. The COMM domain of the Trp synthase-OIA complex is found as a mixture of two conformations. The (3R)-diastereomer of DOA binds about 5-fold more tightly than (3S)-OIA and also forms a mixture of aldimine and quinonoid complexes. DOA forms an additional H-bond between the 3-OH of DOA and βLys-87. l-Trp does not form a covalent complex with the PLP of Trp synthase. However, d-Trp forms a mixture of two external aldimine complexes which differ in the orientation of the α-carboxylate. In one conformation, the α-carboxylate is in the plane of the PLP, while in the other conformation, the α-carboxylate is perpendicular to the PLP plane. These results confirm that the stereochemistry of the transient indolenine quinonoid intermediate in the mechanism of Trp synthase is (3S) and demonstrate the linkage between aldimine and quinonoid reaction intermediates in the β-active site and allosteric communications with the α-active site.