A Study on the Kinetic Mechanism of Apoenzyme Reconstitution from Aerococcus viridans Lactate Oxidase

The preparation of a reconstitutable apoprotein is widely recognized as an important tool for studying the interactions between protein and coenzyme and also for characterizing the coenzyme-binding site of the protein. Here is described the kinetic analysis of the reconstitution of Aerococcus viridans lactate oxidase apoenzyme with FMN and FAD in the presence of substrate. The reconstitution was followed by measuring the increase in catalytic capacity with time. Lactate oxidase activity was easily removed by obtaining its apoenzyme in an acidic saturated ammonium sulphate solution. When the apoenzyme was reconstituted by the addition of FMN or FAD, a marked lag period was observed, after which the system reached a steady state (linear rate). To explain the binding mechanism of the cofactors to the apoenzyme, a kinetic model is proposed, in which the constants, k₃ and k_-3, representing the interaction of apoenzyme with cofactor are considered slow and responsible for the lag in the expression of activity. The affinity of apoenzyme was 51-fold higher for FMN than FAD.     

Isolation and characterization of the transient,luciferase-bound flavin-4a-hydroxide in the bacterial luciferase reaction

Procedures and conditions have been established such that the unstable enzyme-bound flavin intermediate produced in the bacterial luciferase reaction can be isolated as approximately 70% of the flavin product, the remaining being the final product, FMN. The structure of the intermediate is proposed to be that of a luciferase-bound 4a,5-dihydroflavin-4a-hydroxide. The intermediate has a half-life of 33 min at 2°C and decays spontaneously to give H₂O and luciferase-bound FMN with an activation enthalpy of about 120 kJ/mol. It has an absorption spectrum (λ_max = 360 nm) that is consistent with the proposed structure, and a fluorescence emission (λ_max = 485 nm) that matches the bioluminescence emission closely.     

Uncoupling of the substrate monooxygenation and reduced pyridine nucleotide oxidation activities of salicylate hydroxylase by flavins

The NAD(P)H oxidation and substrate monooxygenation activities of Pseudomonas cepacia salicylate hydroxylase can be uncoupled by added flavins. The uncoupling is postulated to result from a reducing equivalent exchange between the hydroxylase-bound FADH₂ and the added flavins, leading to the reduction of the latter species and the regeneration of oxidized holoenzyme without hydroxylating the salicylate substrate. When exogenous FMN was added, the salicylate hydroxylase-catalyzed NAD(P)H oxidation could be coupled to the bacterial bioluminescence reaction, which is specific for fully reduced FMN as a substrate. The quantum yield of the coupled bioluminescence, based on the amount of NADH oxidized independently of salicylate monooxygenation, was determined to be 0.14 correlating closely with the known quantum yield of about 0.17 for reduced FMN in the luciferase-catalyzed bioluminescence reaction. A series of flavin derivatives were tested for their effects on the uncoupling of NAD(P)H oxidation and substrate monooxygenation activities of salicylate hydroxylase. Results indicated that the efficiency for interactions between the bound FADH₂ and free flavins was sensitive to the position of structural modification, size, and charge of the added flavin species, suggesting that the bound FADH₂ was partially exposed to aqueous medium under conditions of actual catalysis.     

Studies on the biosynthesis of cytochrome P-450 in rat liver--a probe with phenobarbital.

The reaction of NAD(P)H:flavin oxidoreductase (flavin reductase) from Photobacterium fischeri is proposed to follow a ping-pong bisubstrate-biproduct mechanism. This is based on a steady-state kinetic analysis of initial velocities and patterns of inhibition by NAD⁺ and AMP. The double reciprocal plots of initial velocities versus concentrations of FMN or NADH show, in both cases, a series of parallel lines. The Michaelis constants for NADH (FMN saturating) and FMN (NADH saturating) are 2.2 and 1.2 × 10^?4m, respectively. The product NAD⁺ has been found to be an inhibitor competitive with FMN but non-competitive with NADH. Using AMP as an inhibitor, noncompetitive inhibition patterns were observed with respect to both NADH and FMN as the varied substrate. In addition, the reductase was not inactivated by treatment with N-ethylmaleimide either alone or in the presence of FMN, but the enzyme was inactivated by N-ethylmaleimide in the presence of NADH. These findings suggest that flavin reductase shuttles between disulfide- and sulfhydryl-containing forms during catalysis.     

Metaphosphate-dependent phosphorylation of riboflavin to FMN by Corynebacterium ammoniagenes

Using the bifunctional FAD synthetase from Corynebacterium ammoniagenes, which has the two sequential activities of flavokinase and FMN adenylyl-transferase in FAD biosynthesis, a method of production of the intermediate FMN without any accumulation of FAD was investigated. Various phosphate polymers having no adenylyl moiety were tested for their ability to phosphorylate riboflavin to FMN, using a crude enzyme from C. ammoniagenes/pKH46, which is an FAD-synthetase-gene-dosed strain. Only metaphosphate, other than ATP, could phosphorylate riboflavin to FMN, but FAD did not accumulate at all. The conditions for the conversion of riboflavin to FMN were optimized. The metaphosphate-dependent phosphorylation reaction required Mg²⁺ as the most effective divalent cation. The best concentrations were 10 mM for MgCl₂ and 3mg/ml for metaphosphate. The riboflavin added to the reaction mixture was almost completely converted into FMN after 6 h incubation in the presence of high concentrations of the enzyme preparation.     

The enthalpy of oxidation of flavin mononucleotide

The oxidation enthalpy of reduced flavin mononucleotide at pH 7.0 in 0.2 m phosphate buffer has been studied by determining the heat associated with the reaction: FMNH₂ + 2 Fe(CN)^?3₆ ? FMN + 2 Fe(CN)^?4₆ + 2 H⁺. (a) (The quinone, semiquinone, and hydroquinone forms of FMN are represented as FMN, FMNH, and FMNH₂, respectively.) Calorimetric experiments were performed in a flow microcalorimeter which was modified to prevent sample contamination by oxygen. The enthalpy observed for reaction (a), after correction for dilution and buffer effects, was ?39.2 ± 0.4 kcal (mole FMNH₂)^?1 at 25 °C. The potential difference, ΔE′, developed by reaction (a) was determined potentiometrically and corresponded to a free energy change, ΔG′, of ?30.3 kcal (mole FMNH₂)^?1. The resulting entropy change, ΔS′, was thus calculated to be ?29.8 e.u. Reaction (a) was also studied at temperatures of 7 °C and 35.5 °C. ΔC_p′ for the reaction was calculated as ?155 ± 18 cal deg^?1 (mole FMNH₂)^?1 at 20 °C. ΔH′ for the reaction (b), FMNH₂ ? FMN + H₂, (b) was calculated as +14.2 ± 0.7 kcal mole^?1 at 25 °C, relative to the enthalpy of the hydrogen electrode being identically equal to zero at all values of pH and temperature. The free energy at pH 7.0 for reaction (b), calculated from the potential was found to be ?9.7 kcal mole^?1, which resulted in an entropy for reaction (b) of 80.2 e.u. A thermal titration of reaction (a) was used to calculate the thermodynamic parameters for the formation of semiquinone dimer according to the reaction FMNH₂ + FMN ? (·FMNH)₂. (c) The free energy, enthalpy, and entropy changes for reaction (c) were estimated to be ?6.1 kcal mole^?1, ?7 kcal mole^?1, and ?3 e.u., respectively.     

Role of neighboring FMN side chains in the modulation of flavin reduction potentials and in the energetics of the FMN:apoprotein interaction in Anabaena flavodoxin

Flavodoxins (Flds) are electron transfer proteins that carry a noncovalently bound flavin mononucleotide molecule (FMN) as a redox active center. A distinguishing feature of these flavoproteins is the dramatic change in the E(sq/rd) reduction potential of the FMN upon binding to the apoprotein (at pH 8.0, from -269 mV when free in solution to -438 mV in Anabaena Fld). In this study, the contribution of three neighboring FMN residues, Thr56, Asn58, and Asn97, and of three negatively charged surface residues, Glu20, Asp65, and Asp96, to modulate the redox properties of FMN upon its binding to the apoprotein has been investigated. Additionally, the role of these residues in the apoflavodoxin:FMN interaction has been analyzed. Concerning the redox potentials, the most noticeable result was obtained for the Thr56Gly mutant. In this Fld variant, the increased accessibility of FMN leads to an increase of +63 mV in the E(sq/rd) value. On the other hand, a correlation between the electrostatic environment of FMN and the E(sq/rd) has been observed. The more positive residues or the less negative residues present in the surroundings of the FMN N(1) atom, then the less negative the value for E(sq/rd). With regard to FMN binding to apoflavodoxin, breaking of hydrophobic interactions between FMN and residues 56, 58, and 97 seems to increase the K(d) values, especially in the Thr56Gly Fld. Such results suggest that the H-bond network in the FMN environment influences the FMN affinity.     

Catalytic properties of the isolated diaphorase fragment of the NAD-reducing [NiFe]-hydrogenase from Ralstonia eutropha

The NAD⁺-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H₂-driven reduction of NAD⁺, as well as reverse electron transfer from NADH to H⁺, in the presence of O₂. It comprises six subunits, HoxHYFUI₂, and incorporates a [NiFe] H⁺/H₂ cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD⁺ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD⁺/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD⁺, respectively. Catalysis in both directions is product inhibited with K _I values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O₂, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis.     

Role of the prosthetic groups of NADPH-cytochrome P450 reductase in 3-hydroxyanthranilamide-catalyzed,NADPH-dependent superoxide anion production

Summary

The role of the prosthetic groups (FAD and FMN) of NADPH-cytochrome P450 reductase (P450 reductase)in 3-hydroxyanthranilamide (3-OH An.Amide)-catalyzed, NADPH-dependent superoxide anion (O₂-) production via the reductase was examined using the native and FMN-depleted preparations of P450 reductase which was partially purified from rat liver microsomes. NADPH-dependent O₂-production by the FMN-depleted preparation was about 10% of that by the native preparation. 3-OH An. Amide-catalyzed, NADPH-dependent O₂-production by the FMN-depleted preparation was less than 10% of that by the native preparation. FMN supplementation returned O₂-production to near normal. We observed the same results for NADPH oxidation and hydrogen peroxide formation. O₂-production, NADPH oxidation, and hydrogen peroxide formation were inhibited by native superoxide dismutase (SOD), but not by boiled, denatured SOD. These results indicate that the prosthetic groups, especially FMN, of P450 reductase play a critical role in 3-OH An.Amide-catalyzed, NADPH-dependent O₂-production via the reductase.     

Properties and significance of a riboflavin-binding hexamerin in the hemolymph of Hyalophora cecropia

A riboflavin-binding hexamerin isolated from pupal hemolymph of Hyalophora cecropia has a native M_r of 510,000, subunit M_r of 85,000, and a 5% carbohydrate content. An intrachain cross-link was confirmed in protease limit digests. Ellman titration confirmed the presence of a sulfhydryl group, which is needed for this linkage. Though Cu²⁺ is known to promote the linkage, heavy metals were not detected in the isolate. Heat denaturation released ligand with the absorbency, fluorescence spectra, and chromatographic behavior of riboflavin. Binding resulted in substantial quenching of the fluorescence of both the isoalloxazine in riboflavin and of aromatic groups in the apoprotein. Kinetic analysis indicated a K_D of 2.5 × 10^?7 M for riboflavin, 1.3 × 10^?7 M for lumiflavin, and greater than 1 × 10^?6 M for FMN and FAD. Over four moles of flavin were bound per mole of hexamerin. The amount of riboflavin in pupal hemolymph is sufficient to occupy only 2–3 of these sites. Riboflavin is also associated with lipophorin and vitellogenin, but the molar ratios after protein isolation were low. On a standard laboratory diet, riboflavin is in great excess, but most of it is apparently excreted before the apoprotein first appears in the hemolymph, just before wandering. The concentration of riboflavin-binding hexamerin rises to 15–30 mg/ml in pupae; relative to other hexamerins, very little is stored in the fat body. All of the apoprotein and 75% of riboflavin disappear from the hemolymph during adult development. An amount of flavin at least equal to that stored in pupal hemolymph is transferred to the eggs formed during this period. © 1994 Wiley-Liss, Inc.     

Purification and Characterization of EDTA Monooxygenase from the EDTA-Degrading Bacterium BNC1

The synthetic chelating agent EDTA can mobilize radionuclides and heavy metals in the environment. Biodegradation of EDTA should reduce this mobilization. Although several bacteria have been reported to mineralize EDTA, little is known about the biochemistry of EDTA degradation. Understanding the biochemistry will facilitate the removal of EDTA from the environment. EDTA-degrading activities were detected in cell extracts of bacterium BNC1 when flavin mononucleotide (FMN), NADH, and O₂ were present. The degradative enzyme system was separated into two different enzymes, EDTA monooxygenase and an FMN reductase. EDTA monooxygenase oxidized EDTA to glyoxylate and ethylenediaminetriacetate (ED3A), with the coconsumption of FMNH₂ and O₂. The FMN reductase provided EDTA monooxygenase with FMNH₂ by reducing FMN with NADH. The FMN reductase was successfully substituted in the assay mixture by other FMN reductases. EDTA monooxygenase was purified to greater than 95% homogeneity and had a single polypeptide with a molecular weight of 45,000. The enzyme oxidized both EDTA complexed with various metal ions and uncomplexed EDTA. The optimal conditions for activity were pH 7.8 and 35°C. K_ms were 34.1 μM for uncomplexed EDTA and 8.5 μM for MgEDTA²⁻; this difference in K_m indicates that the enzyme has greater affinity for MgEDTA²⁻. The enzyme also catalyzed the release of glyoxylate from nitrilotriacetate and diethylenetriaminepentaacetate. EDTA monooxygenase belongs to a small group of FMNH₂-utilizing monooxygenases that attack carbon-nitrogen, carbon-sulfur, and carbon-carbon double bonds.     

Bacterial bioluminescence: absorption and fluorescence characteristics and composition of reaction products of reduced flavin mononucleotide with luciferase and oxygen

The initial reaction products of FMNH₂, oxygen and bacterial luciferase are a flavoprotein, free FMN and hydrogen peroxide. This flavoprotein then adds a mole of oxygen to give a product which either reacts with a long-chain aldehyde to give bioluminescence, or in the absence of aldehyde decays to free enzyme, free FMN and hydrogen peroxide.     

Regulation of nitrate reductase in cyanobacteria. Repression-derepression control of nitrate reductase apoprotein in the cyanobacterium Nostoc muscorum

The repression-derepression control of Nostoc muscorum nitrate reductase was studied with regard to the Mo-cofactor and apoprotein levels. It was found that the synthesis of Mo-cofactor is constitutive but the apoprotein is subject to the repression-derepression control. In NH₄⁺ medium apoprotein synthesis was repressed and in N₂ and NO₃^? media apoprotein synthesis was derepressed. The apoprotein levels were similar in NO₃^? and N₂ media; however, the nitrate reductase activity was lower in N₂ medium due to lower Mo-cofactor activity. The lower Mo-cofactor activity in N₂-fixing conditions as compared to that in non-N₂-fixing conditions was consistent with the earlier view that the Mo-cofactor of nitrate reductase may be a precursor for FeMo-cofactor of nitrogenase.     

The thermodynamics of flavin binding to the apoflavodoxin from Azotobacter vinelandii

A thermodynamic study of the binding of flavins (FMN, FAD, 8-carboxylic acid-riboflavin) to the purified apoflavodoxin from Azotobacter vinelandii has been conducted. The binding of FMN was studied at a number of temperatures (10,15, 20, 25, and 30 °C), pH's (6.0, 7.4, and 9.0), and buffer conditions. The binding of FAD was studied at pH 7.4 and 25 °C under a number of buffer conditions. The binding of 8-carboxylic acid-riboflavin to the apoflavodoxin and the binding of FMN to the dimeric form of the apoflavodoxin were investigated at pH 7.4 and 25 °C. Enthalpies of binding for FMN, FAD, and 8-carboxylic- acid-riboflavin were ?28.3, ?16.6, and ?14.0 kcal mol^?1, respectively. The enthalpy of binding of FMN to the dimeric form of the apoflavodoxin was ?22.2 kcal mol of binding sites^?1. Binding constants of about 10⁸,10⁶, and 10⁶ were obtained for the binding of FMN, FAD, and 8-carboxylic acid-riboflavin, respectively. Using established thermodynamic relationships free energy and entropy changes were calculated. The entropy data indicate that a large degree of ordering of the system occurs upon flavin binding. The pH data suggest that FMN may bind in both the mono-and dianion forms, and that binding doesn't change the pK_a of any functional group in the system. It appears that the phosphate group is probably responsible for approximately half the binding enthalpy observed for the binding of FMN. The temperature-dependence data over the temperature range studied is biphasic, centered at 20 °C, indicating that flavin binding occurs to the protein in two thermodynamic states corresponding to the two heat capacities observed. These findings are used to discuss a model for flavin binding.     

Insights into the role of F26 residue in the FMN: ATP adenylyltransferase activity of Staphylococcus aureus FAD synthetase

The bifunctional flavin adenine dinucleotide synthetase (FADS) synthesizes the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) co-factors essential for the function of flavoproteins. The Staphylococcus aureus FADS (SaFADS) produces FMN from riboflavin (RF) by ATP:riboflavin kinase (RFK) activity at its C-terminal domain. The N-terminal domain converts FMN to FAD under a reducing environment by FMN:ATP adenylyltransferase (FMNAT) activity which is reversible (FAD pyrophosphorylase activity). Herein, we investigated the role of F26 residue of the 24-GFFD-28 motif of SaFADS FMNAT domain, mostly conserved in the reducing agent-dependent FADSs. The steady-state kinetics studies showed changes in the K_m^ATP values for mutants, indicating that the F26 residue is crucial for the FMNAT activity. Further, the FMNAT activity of the F26S mutant was observed to be higher than that of the wild-type SaFADS and its other variants at lower reducing agent concentration. In addition, the FADpp activity was inhibited by an excess of FAD substrate, which was more potent in the mutants. The altered orientation of the F26 side-chain observed in the molecular dynamics analysis suggested its plausible involvement in stabilizing FMN and ATP substrates in their respective binding pockets. Also, the SaFADS ternary complex formed with reduced FMN exhibited significant structural changes in the β4n-β5n and L3n regions compared to the oxidised FMN bound and apo forms of SaFADS. Overall, our data suggests the functional role of F26 residue in the FMNAT domain of SaFADS.     

Identity of aliphatic L- -hydroxyacid oxidase and glycolate oxidase from rat livers

l-α-Hydroxyacid oxidase and glycolate oxidase have been partially purified from rat livers and found to be identical, judging by substrate specificities, K_m values for certain substrates and coenzyme (FMN), activation energy, inhibition rates by various reagents and pH optimum. K_m values are as follows; glycolate, 2.4 × 10^?4m; l-α-hydroxyisocaproate, 1.26 × 10^?3; glyoxylate, 1.41 × 10^?4m; and FMN, 1.13 × 10^?6m. K_m values for glycolate and FMN are one-tenth and one-twentieth the literature values for hepatic glycolate oxidase. Sucrose density gradient centrifugation establishes that this enzyme is located in hepatic peroxisomes.     

NMR-derived topology of a GFP-photoprotein energy transfer complex

Förster resonance energy transfer within a protein-protein complex has previously been invoked to explain emission spectral modulation observed in several bioluminescence systems. Here we present a spatial structure of a complex of the Ca²⁺-regulated photoprotein clytin with its green-fluorescent protein (cgGFP) from the jellyfish Clytia gregaria, and show that it accounts for the bioluminescence properties of this system in vitro. We adopted an indirect approach of combining x-ray crystallography determined structures of the separate proteins, NMR spectroscopy, computational docking, and mutagenesis. Heteronuclear NMR spectroscopy using variously ¹⁵N,¹³C,²H-enriched proteins enabled assignment of backbone resonances of more than 94% of the residues of both proteins. In a mixture of the two proteins at millimolar concentrations, complexation was inferred from perturbations of certain ¹H-¹⁵N HSQC-resonances, which could be mapped to those residues involved at the interaction site. A docking computation using HADDOCK was employed constrained by the sites of interaction, to deduce an overall spatial structure of the complex. Contacts within the clytin-cgGFP complex and electrostatic complementarity of interaction surfaces argued for a weak protein-protein complex. A weak affinity was also observed by isothermal titration calorimetry (K_D = 0.9 mm). Mutation of clytin residues located at the interaction site reduced the degree of protein-protein association concomitant with a loss of effectiveness of cgGFP in color-shifting the bioluminescence. It is suggested that this clytin-cgGFP structure corresponds to the transient complex previously postulated to account for the energy transfer effect of GFP in the bioluminescence of aequorin or Renilla luciferase.     

The enthalpy of oxidation of flavin mononucleotide: Temperature dependence of in vitro bacterial luciferase bioluminescence

The enthalpy of the bioluminescent reaction

$\text{FMNH}{}_2\text{+ RCHO + O}{}_2\text{→}\text{luciferase}\text{FMN + RCOO + H}{}_3\text{O}{}^{\mathrm{+}}\text{+ hv}$

has been studied by direct calorimetric methods. Bacterial luciferase, isolated from Beneckea harveyi (formerly strain MAV) has been used to catalyze the oxidation of reduced flavin mononucleotide (FMNH₂) and a long chain aliphatic aldehyde (dodecanal, RCHO) by molecular oxygen to give the indicated products and blue-green light. The enthalpy measured for this process was found to be ΔH_L = ?338.9 k.J (mol FMN)^?1 (?81.0 kcal) at 25.00 °C and ?402.9 kJ (mol FMN)^?1 (?96.3 kcal) at 7.00 °C. Calculations based on redox electrode potentials indicate a corresponding value of the free energy change, ΔG_L = ?464.8 kJ (mol FMN)^?1 (?111.1 kcal), at 25 °C. Measurements were performed in 0.15 m phosphate buffer, pH 7.0 and the values were arrived at by correcting the observed heats for the heat associated with the autoxidation process: FMNH₂ + O₂ ? FMN + H₂O₂; ΔH_D = ?158.5 kJ (mol FMN)^?1 (?37.8). These data and a detailed thermodynamic analysis have demonstrated the need for two parameters, referred to as the intrinsic free energy, ΔG₁, and intrinsic enthalpy, ΔH₁, which are functionally defined by the relations ΔG_I = ΔG_L ? uhvΔH_I = ΔH_L ? uhv, where u is the quantum yield of the reaction expressed in einsteins mole^?1.These parameters reflect the thermochemistry of the bioluminescent reaction corrected for emitted photons. Thus, they are useful for comparing the thermochemistry of a chemiluminescent process. Their values for the bacterial luciferase system at 25 °C and pH 7.0 are ?391.6 and ?266.9 kJ (mol FMN)^?1 (?93.6 and ?63.8 kcal), respectively, assuming a value of 0.3 for the quantum yield. The calorimetric data also suggest the existence of a long-lived species which persists after photon emission.     

Two Lysine Residues in the Bacterial Luciferase Mobile Loop Stabilize Reaction Intermediates

Bacterial luciferase catalyzes the reaction of FMNH₂, O₂, and a long chain aliphatic aldehyde, yielding FMN, carboxylic acid, and blue-green light. The most conserved contiguous region of the primary sequence corresponds to a crystallographically disordered loop adjacent to the active center (Fisher, A. J., Raushel, F. M., Baldwin, T. O., and Rayment, I. (1995) Biochemistry 34, 6581–6586; Fisher, A. J., Thompson, T. B., Thoden, J. B., Baldwin, T. O., and Rayment, I. (1996) J. Biol. Chem. 271, 21956–21968). Deletion of the mobile loop does not alter the chemistry of the reaction but decreases the total quantum yield of bioluminescence by 2 orders of magnitude (Sparks, J. M., and Baldwin, T. O. (2001) Biochemistry 40, 15436–15443). In this study, we attempt to localize the loss of activity observed in the loop deletion mutant to individual residues in the mobile loop. Using alanine mutagenesis, the effects of substitution at 15 of the 29 mobile loop residues were examined. Nine of the point mutants had reduced activity in vivo. Two mutations, K283A and K286A, resulted in a loss in quantum yield comparable with that of the loop deletion mutant. The bioluminescence emission spectrum of both mutants was normal, and both yielded the carboxylic acid chemical product at the same efficiency as the wild-type enzyme. Substitution of Lys²⁸³ with alanine resulted in destabilization of intermediate II, whereas mutation of Lys²⁸⁶ had an increase in exposure of reaction intermediates to a dynamic quencher. Based on a model of the enzyme-reduced flavin complex, the two critical lysine residues are adjacent to the quininoidal edge of the isoalloxazine.     

Hydrogen peroxide production and the release of carbon dioxide during glycollate oxidation in leaf peroxisomes

Summary The rate at which H₂O₂ becomes available during glycollate oxidation for further oxidation reactions, especially that of glyoxylate to formate and CO₂, in peroxisomes from spinach-beet (Beta vulgaris L., var. vulgaris) leaves has been determined by measuring O₂ uptake in the presence and absence of added catalase. The rates observed under air and pure O₂ were sufficient to account for the ¹⁴CO₂ released from [l-¹⁴C]glycollate under these conditions; the two reactions showed similar characteristics. In the course of the reaction, a fall in catalase activity was observed concomitant with an increase in ¹⁴CO₂ release. There is no evidence that catalase was disproportionately lost from the peroxisomes during isolation, and it is argued that the CO₂ release observed contributes to the photorespiratory CO₂ loss in intact leaves.Abbreviations DCPIP 2,6-dichlorophenolindophenol - FMN Flavin mononucleotide