Hydrogen peroxide probes directed to different cellular compartments

Background

Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells.

Principal Findings

Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed in each of these compartments, consistent with a low peroxide tone in mammalian cells. In contrast, HyPer was mostly oxidized in the endoplasmic reticulum. Using this system, we characterized control of hydrogen peroxide in various cell systems, such as cells deficient in thioredoxin reductase, sulfhydryl oxidases or subjected to selenium deficiency. Generation of hydrogen peroxide could also be monitored in various compartments following signaling events.

Conclusions

We found that HyPer can be used as a valuable tool to monitor hydrogen peroxide generated in different cellular compartments. The data also show that hydrogen peroxide generated in one compartment could translocate to other compartments. Our data provide information on compartmentalization, dynamics and homeostatic control of hydrogen peroxide in mammalian cells.     

Redox responses of Arabidopsis thaliana to the green peach aphid,Myzus persicae

The green peach aphid (Myzus persicae) is a phloem-feeding insect that causes economic damage on a wide array of crops. Using a luminol-based assay, a superoxide-responsive reporter gene (Zat12::luciferase), and a probe specific to hydrogen peroxide (HyPer), we demonstrated that this aphid induces accumulation of reactive oxygen species (ROS) in Arabidopsis thaliana. Similar to the apoplastic oxidative burst induced by pathogens, this response to aphids was rapid and transient, with two peaks occurring within 1 and 4 hr after infestation. Aphid infestation also induced an oxidative response in the cytosol and peroxisomes, as measured using a redox-sensitive variant of green fluorescent protein (roGFP2). This intracellular response began within minutes of infestation but persisted 20 hr or more after inoculation, and the response of the peroxisomes appeared stronger than the response in the cytosol. Our results suggest that the oxidative response to aphids involves both apoplastic and intracellular sources of ROS, including ROS generation in the peroxisomes, and these different sources of ROS may potentially differ in their impacts on host suitability for aphids.     

Kinetics and mechanisms of catalase in peroxisomes of the mitochondrial fraction

1. The primary intermediate of catalase and hydrogen peroxide was identified and investigated in peroxisome-rich mitochondrial fractions of rat liver. On the basis of kinetic constants determined in vitro, it is possible to calculate with reasonable precision the molecular statistics of catalase action in the peroxisomes. 2. The endogenous hydrogen peroxide generation is adequate to sustain a concentration of the catalase intermediate (p(m)/e) of 60-70% of the hydrogen peroxide saturation value. Total amount of catalase corresponds to 0.12-0.15nmol of haem iron/mg of protein. In State 1 the rate of hydrogen peroxide generation corresponds to 0.9nmol/min per mg of protein or 5% of the mitochondrial respiratory rate in State 4. 3. Partial saturation of the catalase intermediate with hydrogen peroxide (p(m)/e) in the mitochondrial fraction suggests its significant peroxidatic activity towards its endogenous hydrogen donor. A variation of this value (p(m)/e) from 0.3 in State 4 to 0 under anaerobic conditions is observed. 4. For a particular preparation the hydrogen peroxide generation rate in the substrate-supplemented State 4 corresponds to 0.17s(-1) (eqn. 6), the hydrogen peroxide concentration to 2.5nm and the hydrogen-donor concentration (in terms of ethanol) to 0.12mm. The reaction is 70% peroxidatic and 30% catalatic. 5. A co-ordinated production of both oxidizing and reducing substrates for catalase in the mitochondrial fraction is suggested by a 2.2-fold increase of hydrogen peroxide generation and a threefold increase in hydrogen-donor generation in the State 1 to State 4 transition. 6. Additional hydrogen peroxide generation provided by the urate oxidase system of peroxisomes (8-12nmol of uric acid oxidized/min per mg of protein) permits saturation of the catalase with hydrogen peroxide to haem occupancy of 40% compared with values of 36% for a purified rat liver catalase ofk(1)=1.7x10(7)m(-1).s(-1) and k'(4)=2.6x10(7)m(-1). s(-1)(Chance, Greenstein & Roughton, 1952). 7. The turnover of the catalase ethyl hydrogen peroxide intermediate (k'(3)) in the peroxisomes is initially very rapid since endogenous hydrogen peroxide acts as a hydrogen donor. k'(3) decreases fivefold in the uncoupled state of the mitochondria.     

The H(2)O(2)-sensitive HyPer protein targeted to the endoplasmic reticulum as a mirror of the oxidizing thiol-disulfide milieu

Oxidative protein folding in the endoplasmic reticulum (ER) is associated with the formation of native disulfide bonds, which inevitably results in the formation of hydrogen peroxide (H(2)O(2)). Particularly in pancreatic β-cells with their high secretory activity and extremely low antioxidant capacity, the H(2)O(2) molecules generated during oxidative protein folding could represent a significant oxidative burden. Therefore this study was conducted to elucidate the H(2)O(2) generation during disulfide bond formation in insulin-producing RINm5F cells by targeting and specifically expressing the H(2)O(2)-sensitive biosensor HyPer in the ER (ER-HyPer). In addition the influence of overexpression of the H(2)O(2)-metabolizing ER-resident peroxiredoxin IV (PRDXIV) on H(2)O(2) levels was examined. The ER-HyPer fluorescent protein was completely oxidized, whereas HyPer expressed in cytosol, peroxisomes, and mitochondria was prevalently in the reduced state, indicating a high basal H(2)O(2) concentration in the ER lumen. These results could also be confirmed in non-insulin-producing COS-7 cells. Overexpression of PRDXIV in RINm5F cells effectively protected against H(2)O(2)-mediated toxicity; however, it did not affect the fluorescence signal of ER-HyPer. Moreover, the inhibition of de novo protein synthesis and the associated H(2)O(2) generation by cycloheximide had no influence on the ER-HyPer redox state. Taken together, these findings strongly suggest that the H(2)O(2)-sensitive biosensor reflects exclusively the oxidative milieu in the ER and not the H(2)O(2) concentration in the ER lumen.     

Peroxisomes as a source of reactive oxygen species and nitric oxide signal molecules in plant cells

The important role of plant peroxisomes in a variety of metabolic reactions such as photorespiration, fatty acid beta-oxidation, the glyoxylate cycle and generation-degradation of hydrogen peroxide is well known. In recent years, the presence of a novel group of enzymes, mainly involved in the metabolism of oxygen free-radicals, has been shown in peroxisomes. In addition to hydrogen peroxide, peroxisomes can generate superoxide-radicals and nitric oxide, which are known cellular messengers with a variety of physiological roles in intra- and inter-cellular communication. Nitric oxide and hydrogen peroxide can permeate the peroxisomal membrane and superoxide radicals can be produced on the cytosolic side of the membrane. The signal molecule-generating capacity of peroxisomes can have important implications for cellular metabolism in plants, particularly under biotic and abiotic stress.     

Feasibility of simultaneous measurement of cytosolic calcium and hydrogen peroxide in vascular smooth muscle cells

Interplay between calcium ions (Ca²⁺) and reactive oxygen species (ROS) delicately controls diverse pathophysiological functions of vascular smooth muscle cells (VSMCs). However, details of the Ca²⁺ and ROS signaling network have been hindered by the absence of a method for dual measurement of Ca²⁺ and ROS. Here, a real-time monitoring system for Ca²⁺ and ROS was established using a genetically encoded hydrogen peroxide indicator, HyPer, and a ratiometric Ca²⁺ indicator, fura-2. For the simultaneous detection of fura-2 and HyPer signals, 540 nm emission filter and 500 nm∼ dichroic beamsplitter were combined with conventional exciters. The wide excitation spectrum of HyPer resulted in marginal cross-contamination with fura-2 signal. However, physiological Ca²⁺ transient and hydrogen peroxide were practically measurable in HyPer-expressing, fura-2-loaded VSMCs. Indeed, distinct Ca²⁺ and ROS signals could be successfully detected in serotonin-stimulated VSMCs. The system established in this study is applicable to studies of crosstalk between Ca²⁺ and ROS. [BMB Reports 2013; 46(12): 600-605]     

Genetically encoded fluorescent indicator for intracellular hydrogen peroxide

We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H(2)O(2)) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H(2)O(2)-sensing protein, OxyR. Using HyPer we monitored H(2)O(2) production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/TRAIL. We found that an increase in H(2)O(2) occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (DeltaPsi) and a change in cell shape. We also observed local bursts in mitochondrial H(2)O(2) production during DeltaPsi oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H(2)O(2) increase upon physiological stimulation. Using HyPer we detected temporal increase in H(2)O(2) in the cytoplasm of PC-12 cells stimulated with nerve growth factor.     

NOX4-dependent Hydrogen peroxide promotes shear stress-induced SHP2 sulfenylation and eNOS activation

Laminar shear stress (LSS) triggers signals that ultimately result in atheroprotection and vasodilatation. Early responses are related to the activation of specific signaling cascades. We investigated the participation of redox-mediated modifications and in particular the role of hydrogen peroxide (H₂O₂) in the sulfenylation of redox-sensitive phosphatases. Exposure of vascular endothelial cells to short periods of LSS (12 dyn/cm²) resulted in the generation of superoxide radical anion as detected by the formation of 2-hydroxyethidium by HPLC and its subsequent conversion to H₂O₂, which was corroborated by the increase in the fluorescence of the specific peroxide sensor HyPer. By using biotinylated dimedone we detected increased total protein sulfenylation in the bovine proteome, which was dependent on NADPH oxidase 4 (NOX4)-mediated generation of peroxide. Mass spectrometry analysis allowed us to identify the phosphatase SHP2 as a protein susceptible to sulfenylation under LSS. Given the dependence of FAK activity on SHP2 function, we explored the role of FAK under LSS conditions. FAK activation and subsequent endothelial NO synthase (eNOS) phosphorylation were promoted by LSS and both processes were dependent on NOX4, as demonstrated in lung endothelial cells isolated from NOX4-null mice. These results support the idea that LSS elicits redox-sensitive signal transduction responses involving NOX4-dependent generation of hydrogen peroxide, SHP2 sulfenylation, and ulterior FAK-mediated eNOS activation.     

Light-induced intracellular hydrogen peroxide generation through genetically encoded photosensitizer KillerRed-SOD1

Abstract

Hydrogen peroxide (H₂O₂) plays an important role in various biological processes in numerous organisms. Depending on the concentration and the distribution within the cell, it can act as stressor or redox signalling molecule. To analyse the effects of H₂O₂ and its diffusion within the cell we developed the new genetically encoded photosensitizer KillerRed-SOD1 which enables a light-induced spatially and temporally controlled generation of H₂O₂ in living cells. The KillerRed-SOD1 is a fusion protein of the photosensitizer KillerRed (KR) and the cytosolic superoxide dismutase isoform 1 (SOD1) connected by a helix-forming peptide linker. Light irradiation at a wavelength of 560?nm induced superoxide radical formation at the KR domain which was transformed to H₂O₂ at the SOD1 domain. H₂O₂ was specifically detected under live cell conditions using the fluorescent sensor protein HyPer. Genetically encoded photosensitizers have the advantage that appropriate tag sequences can determine the localisation of the protein within the cell. Herein, it was exemplarily shown that the peroxisomal targeting sequence 1 directed the photosensitizer KR-SOD1 to the peroxisomes and enabled H₂O₂ formation specifically in these organelles. In summary, with the photosensitizer KR-SOD1 a new valuable tool was established which allows a controlled intracellular H₂O₂ generation for the analysis of H₂O₂ effects on a subcellular level.     

H2O2 in plant peroxisomes: an in vivo analysis uncovers a Ca2+‐dependent scavenging system

Oxidative stress is a major challenge for all cells living in an oxygen‐based world. Among reactive oxygen species, H₂O₂, is a well known toxic molecule and, nowadays, considered a specific component of several signalling pathways. In order to gain insight into the roles played by H₂O₂ in plant cells, it is necessary to have a reliable, specific and non‐invasive methodology for its in vivo detection. Hence, the genetically encoded H₂O₂ sensor HyPer was expressed in plant cells in different subcellular compartments such as cytoplasm and peroxisomes. Moreover, with the use of the new green fluorescent protein (GFP)‐based Cameleon Ca²⁺ indicator, D3cpv–KVK–SKL, targeted to peroxisomes, we demonstrated that the induction of cytoplasmic Ca²⁺ increase is followed by Ca²⁺ rise in the peroxisomal lumen. The analyses of HyPer fluorescence ratios were performed in leaf peroxisomes of tobacco and pre‐ and post‐bolting Arabidopsis plants. These analyses allowed us to demonstrate that an intraperoxisomal Ca²⁺ rise in vivo stimulates catalase activity, increasing peroxisomal H₂O₂ scavenging efficiency.     

Plant autophagy is responsible for peroxisomal transition and plays an important role in the maintenance of peroxisomal quality

In photosynthetic cells, a large amount of hydrogen peroxide is produced in peroxisomes through photorespiration, which is a metabolic pathway related to photosynthesis. Hydrogen peroxide, a reactive oxygen species, oxidizes peroxisomal proteins and membrane lipids, resulting in a decrease in peroxisomal quality. We demonstrate that the autophagic system is responsible for the elimination of oxidized peroxisomes in plant. We isolated Arabidopsis mutants that accumulated oxidized peroxisomes, which formed large aggregates. We revealed that these mutants were defective in autophagy-related (ATG) genes and that the aggregated peroxisomes were selectively targeted by the autophagic machinery. These findings suggest that autophagy plays an important role in the quality control of peroxisomes by the selective degradation of oxidized peroxisomes. In addition, the results suggest that autophagy is also responsible for the functional transition of glyoxysomes to leaf peroxisomes.     

Plant autophagy is responsible for peroxisomal transition and plays an important role in the maintenance of peroxisomal quality

In photosynthetic cells, a large amount of hydrogen peroxide is produced in peroxisomes through photorespiration, which is a metabolic pathway related to photosynthesis. Hydrogen peroxide, a reactive oxygen species, oxidizes peroxisomal proteins and membrane lipids, resulting in a decrease in peroxisomal quality. We demonstrate that the autophagic system is responsible for the elimination of oxidized peroxisomes in plant. We isolated Arabidopsis mutants that accumulated oxidized peroxisomes, which formed large aggregates. We revealed that these mutants were defective in autophagy-related (ATG) genes and that the aggregated peroxisomes were selectively targeted by the autophagic machinery. These findings suggest that autophagy plays an important role in the quality control of peroxisomes by the selective degradation of oxidized peroxisomes. In addition, the results suggest that autophagy is also responsible for the functional transition of glyoxysomes to leaf peroxisomes.     

Mammalian peroxisomes and reactive oxygen species

The central role of peroxisomes in the generation and scavenging of hydrogen peroxide has been well known ever since their discovery almost four decades ago. Recent studies have revealed their involvement in metabolism of oxygen free radicals and nitric oxide that have important functions in intra- and intercellular signaling. The analysis of the role of mammalian peroxisomes in a variety of physiological and pathological processes involving reactive oxygen species (ROS) is the subject of this review. The general characteristics of peroxisomes and their enzymes involved in the metabolism of ROS are briefly reviewed. An expansion of the peroxisomal compartment with proliferation of tubular peroxisomes is observed in cells exposed to UV irradiation and various oxidants and is apparently accompanied by upregulation of PEX genes. Significant reduction of peroxisomes and their enzymes is observed in inflammatory processes including infections, ischemia-reperfusion injury, and allograft rejection and seems to be related to the suppressive effect of tumor necrosis factor- on peroxisome function and peroxisome proliferator activated receptor-. Xenobiotic-induced proliferation of peroxisomes in rodents is accompanied by the formation of hepatic tumors, and evidently the imbalance in generation and decomposition of ROS plays an important role in this process. In PEX5^–/– knockout mice lacking functional peroxisomes severe alterations of mitochondria in various organs are observed which seem to be due to a generalized increase in oxidative stress confirming the important role of peroxisomes in homeostasis of ROS and the implications of its disturbances for cell pathology.     

Dynamic measurements of mitochondrial hydrogen peroxide concentration and glutathione redox state in rat pancreatic β-cells using ratiometric fluorescent proteins: confounding effects of pH with HyPer but not roGFP1

Using the ROS (reactive oxygen species)-sensitive fluorescent dyes dichlorodihydrofluorescein and dihydroethidine, previous studies yielded opposite results about the glucose regulation of oxidative stress in insulin-secreting pancreatic β-cells. In the present paper, we used the ratiometric fluorescent proteins HyPer and roGFP1 (redox-sensitive green fluorescent protein 1) targeted to mitochondria [mt-HyPer (mitochondrial HyPer)/mt-roGFP1 (mitochondrial roGFP1)] to monitor glucose-induced changes in mitochondrial hydrogen peroxide concentration and glutathione redox state in adenovirus-infected rat islet cell clusters. Because of the reported pH sensitivity of HyPer, the results were compared with those obtained with the mitochondrial pH sensors mt-AlpHi and mt-SypHer. The fluorescence ratio of the mitochondrial probes slowly decreased (mt-HyPer) or increased (mt-roGFP1) in the presence of 10 mmol/l glucose. Besides its expected sensitivity to H2O2, mt-HyPer was also highly pH sensitive. In agreement, changes in mitochondrial metabolism similarly affected mt-HyPer, mt-AlpHi and mt-SypHer fluorescence signals. In contrast, the mt-roGFP1 fluorescence ratio was only slightly affected by pH and reversibly increased when glucose was lowered from 10 to 2 mmol/l. This increase was abrogated by the catalytic antioxidant Mn(III) tetrakis (4-benzoic acid) porphyrin but not by N-acetyl-L-cysteine. In conclusion, due to its pH sensitivity, mt-HyPer is not a reliable indicator of mitochondrial H2O2 in β-cells. In contrast, the mt-roGFP1 fluorescence ratio monitors changes in β-cell mitochondrial glutathione redox state with little interference from pH changes. Our results also show that glucose acutely decreases rather than increases mitochondrial thiol oxidation in rat β-cells.     

Glutamate-induced metabolic changes influence the cytoplasmic redox state of hippocampal neurons

Brain cell metabolism is intimately associated with intracellular oxidation–reduction (redox) balance. Glutamatergic transmission is accompanied with changes in substrate preference in neurons. Therefore, we studied cytoplasmatic redox changes in hippocampal neurons in culture exposed to glutamate. Neurons were transfected with HyPer, a genetically encoded redox biosensor for hydrogen peroxide which allows real-time imaging of the redox state. The rate of fluorescence decay, corresponding to the reduction of the biosensor was found to be augmented by low doses of glutamate (10 μM) as well as by pharmacological stimulation of NMDA glutamate receptors. Acute chelation of extracellular Ca²⁺ abolished the glutamate-induced effect observed on HyPer fluorescence. Additional experiments indicated that mitochondrial function and hence energetic substrate availability commands the redox state of neurons and is required for the glutamate effect observed on the biosensor signal. Furthermore, our results implicated astrocytic metabolism in the changes of neuronal redox state observed with glutamate.     

Highly Oxidized Peroxisomes Are Selectively Degraded via Autophagy in Arabidopsis

The positioning of peroxisomes in a cell is a regulated process that is closely associated with their functions. Using this feature of the peroxisomal positioning as a criterion, we identified three Arabidopsis thaliana mutants (peroxisome unusual positioning1 [peup1], peup2, and peup4) that contain aggregated peroxisomes. We found that the PEUP1, PEUP2, and PEUP4 were identical to Autophagy-related2 (ATG2), ATG18a, and ATG7, respectively, which are involved in the autophagic system. The number of peroxisomes was increased and the peroxisomal proteins were highly accumulated in the peup1 mutant, suggesting that peroxisome degradation by autophagy (pexophagy) is deficient in the peup1 mutant. These aggregated peroxisomes contained high levels of inactive catalase and were more oxidative than those of the wild type, indicating that peroxisome aggregates comprise damaged peroxisomes. In addition, peroxisome aggregation was induced in wild-type plants by exogenous application of hydrogen peroxide. The cat2 mutant also contained peroxisome aggregates. These findings demonstrate that hydrogen peroxide as a result of catalase inactivation is the inducer of peroxisome aggregation. Furthermore, an autophagosome marker, ATG8, frequently colocalized with peroxisome aggregates, indicating that peroxisomes damaged by hydrogen peroxide are selectively degraded by autophagy in the wild type. Our data provide evidence that autophagy is crucial for quality control mechanisms for peroxisomes in Arabidopsis.     

Significance of plant sulfite oxidase

Sulfite oxidizing activities are known since years in animals, microorganisms, and also plants. Among plants, the only enzyme well characterized on molecular and biochemical level is the molybdoenzyme sulfite oxidase (SO). It oxidizes sulfite using molecular oxygen as electron acceptor, leading to the production of sulfate and hydrogen peroxide. The latter reaction product seems to be the reason why plant SO is localized in peroxisomes, because peroxisomal catalase is able to decompose hydrogen peroxide. On the other hand, we have indications for an additional reaction taking place in peroxisomes: sulfite can be nonenzymatically oxidized by hydrogen peroxide. This will promote the detoxification of hydrogen peroxide especially in the case of high amounts of sulfite. Hence we assume that SO could possibly serve as "safety valve" for detoxifying excess amounts of sulfite and protecting the cell from sulfitolysis. Supportive evidence for this assumption comes from experiments where we fumigated transgenic poplar plants overexpressing ARABIDOPSIS SO with SO(2) gas. In this paper, we try to explain sulfite oxidation in its co-regulation with sulfate assimilation and summarize other sulfite oxidizing activities described in plants. Finally we discuss the importance of sulfite detoxification in plants.     

Protection of insulin-producing cells against toxicity of dexamethasone by catalase overexpression

Pancreatic β cells are very sensitive to reactive oxygen species (ROS) and this might play an important role in β cell death in diabetes. Dexamethasone is a synthetic diabetogenic glucocorticoid, which impairs pancreatic β cell function. Therefore we investigated the toxicity of dexamethasone in RINm5F insulin-producing cells and its dependence on the expression level of the antioxidant enzyme catalase, which inactivates hydrogen peroxide. This was correlated with oxidative stress and cell death. An increased generation of ROS was observed in dexamethasone-treated cells together with an increase in caspase-3 activity and apoptosis rate. Interestingly, exposure to dexamethasone increased the cytosolic superoxide dismutase Cu/ZnSOD protein expression and activity, whereas the mitochondrial MnSOD isoform was not affected by the glucocorticoid. Catalase overexpression in insulin-producing cells prevented all the cytotoxic effects of dexamethasone. In conclusion, dexamethasone-induced cell death in insulin-producing cells is ROS mediated. Increased levels of expression and activity of the Cu/ZnSOD might favor the generation of hydrogen peroxide in dexamethasone-treated cells. Increased ROS scavenging capacity in insulin-producing cells, through overexpression of catalase, prevents a deleterious increase in hydrogen peroxide generation and thus prevents dexamethasone-induced apoptosis.     

Expression of active spinach glycolate oxidase in Aspergillus nidulans

The biocatalytic production of glyoxylic acid from glycolic acid requires two enzymes: glycolate oxidase, which catalyzes the oxidation of glycolic acid by oxygen to produce glyoxylic acid and hydrogen peroxide, and catalase, which decomposes the byproduct hydrogen peroxide. As an alternative to isolation from the leaf peroxisomes of spinach, glycolate oxidase has now been cloned and expressed in transformants of Aspergillus nidulans T580 at levels ranging from 1.7 to 36 IU/g dry wt. cells. The glycolate oxidase of transformant strain T17 comprises ca. 1.9% of total cell protein and is expressed at near 100% activity. (c) 1996 John Wiley & Sons, Inc.     

Preparation of Labeled Casein fron Goat Milk after Lysine-14C Injection

Hydrogen peroxide in methylotrophic yeasts can be metabolized in at least two distinct ways. Addition of exogenous hydrogen peroxide removes the dependance of catalase on endogenously-produced hydrogen peroxide resulting enhanced rates of alcohol oxidation. Exogenous hydrogen peroxide is also efficiently degraded by cytochrome c peroxidase (CCP), a competitive reaction which does not result in enhanced alcohol oxidation. To overcome the influence of cytochrome c peroxidase, artificial peroxisomes were prepared by coimmobilization of alcohol oxidase and catalase. These artificial peroxisomes mimic the peroxide-induced rate enhancement observed with whole cells.