o-Diphenol: Oxygen oxidoreductase from leaves of sugar cane

A non-particulate o-diphenol: O₂ oxidoreductase (phenolase) has been isolated from leaves of sugar cane. Gel filtration produced two fractions MW 32000 and 130000. The preferred substrate was chlorogenic acid. Other o-diphenols (caffeic acid, catechol, pyrogallol, dihydroxyphenylalanine) all of which were slowly oxidized when tested alone, increased the rates of O₂ consumption obtained with catalytic amounts of chlorogenic acid. Both enzyme fractions were inhibited by thiols; thioglycollate, which acted in a non-competitive manner, was most effective.     

Exploring the catalytic potential of the 3-His mononuclear nonheme Fe(II) center: discovery and characterization of an unprecedented maltol cleavage activity

Mononuclear nonheme iron enzymes (MNHEs) catalyze a range of very diverse reactions in O₂ metabolism, but they share a common principle active-site organization. To investigate a putative catalytic promiscuity of these enzymatic metal centers, we studied the reactivity of the 3-His ligated metal center of diketone cleaving enzyme (Dke1) toward non-native substrates, with a focus on alternative O₂ dependent reactions. From a screening approach, which aims at eliminating steric factors by including minimal substrate-substructures, three alternative, ‘non-β-dicarbonyl-cleavage’ reactions are identified, among them an unprecedented oxygenation of maltol. Maltol cleavage is characterized by steady state and fast kinetic measurements and shows an O₂ concentration dependent rate determining step k_cat/K_M(O₂) of 0.3 mM^− 1 s^− 1 and a strict coupling of O₂ reduction and substrate oxidation. Furthermore, the catalytic potential of the 3-His metal center for O₂ dependent catechol ring-cleavage and phenylpyruvate oxidation (PP) is demonstrated.     

Properties of polyphenol oxidase from tubers of the yam Dioscorea bulbifera

The subunit MW of Dioscorea bulbifera polyphenol oxidase (MW 115 000 ± 2000) determined by SDS-PAGE is ca. 31 000 indicating that the enzyme is an oligomeric protein with four subunits. K_i values of various inhibitors and their modes of inhibition have been determined with catechol and pyrogallol as substrates. p-Nitrophenol, p-cresol, quinoline and resorcinol are competitive inhibitors of catechol binding while only orcinol and p-nitrophenol behave in the same way towards pyrogallol as substrate. From the effect of pH on V_max, groups with pK values ca. 4.7 and 6.8 have been identified to be involved in catalytic activity. The Arrhenius activation energy (E_a) at pH 4.0 is 8.9 kcal/mol between 40–65°. At pH 7.0, the value is 22.1 kcal/mol between 40 and 60°. The enthalpies (ΔH) at pH 4.0 and pH 7.0 are 2.3 kcal/mol and 32.4 kcal/mol respectively. The results are discussed considering the conformational changes of the enzyme during substrate binding.     

Availability of O2 as a Substrate in the Cytoplasm of Bacteria under Aerobic and Microaerobic Conditions

The growth rates of Pseudomonas putida KT2442 and mt-2 on benzoate, 4-hydroxybenzoate, or 4-methylbenzoate showed an exponential decrease with decreasing oxygen tensions (partial O₂ tension [pO₂] values). The oxygen tensions resulting in half-maximal growth rates were in the range of 7 to 8 mbar of O₂ (corresponding to 7 to 8 μM O₂) (1 bar = 10⁵ Pa) for aromatic compounds, compared to 1 to 2 mbar for nonaromatic compounds like glucose or succinate. The decrease in the growth rates coincided with excretion of catechol or protocatechuate, suggesting that the activity of the corresponding oxygenases became limiting. The experiments directly establish that under aerobic and microaerobic conditions (about 10 mbar of O₂), the diffusion of O₂ into the cytoplasm occurs at high rates sufficient for catabolic processes. This is in agreement with calculated O₂ diffusion rates. Below 10 mbar of O₂, oxygen became limiting for the oxygenases, probably due to their high K_m values, but the diffusion of O₂ into the cytoplasm presumably should be sufficiently rapid to maintain ambient oxygen concentrations at oxygen tensions as low as 1 mbar of O₂. The consequences of this finding for the availability of O₂ as a substrate or as a regulatory signal in the cytoplasm of bacterial cells are discussed.     

Inhibition of human catechol-O-methyltransferase (COMT)-mediated O-methylation of catechol estrogens by major polyphenolic components present in coffee

In the present study, we investigated the inhibitory effect of three catechol-containing coffee polyphenols, chlorogenic acid, caffeic acid and caffeic acid phenethyl ester (CAPE), on the O-methylation of 2- and 4-hydroxyestradiol (2-OH-E₂ and 4-OH-E₂, respectively) catalyzed by the cytosolic catechol-O-methyltransferase (COMT) isolated from human liver and placenta. When human liver COMT was used as the enzyme, chlorogenic acid and caffeic acid each inhibited the O-methylation of 2-OH-E₂ in a concentration-dependent manner, with IC₅₀ values of 1.3–1.4 and 6.3–12.5 μM, respectively, and they also inhibited the O-methylation of 4-OH-E₂, with IC₅₀ values of 0.7–0.8 and 1.3–3.1 μM, respectively. Similar inhibition pattern was seen with human placental COMT preparation. CAPE had a comparable effect as caffeic acid for inhibiting the O-methylation of 2-OH-E₂, but it exerted a weaker inhibition of the O-methylation of 4-OH-E₂. Enzyme kinetic analyses showed that chlorogenic acid and caffeic acid inhibited the human liver and placental COMT-mediated O-methylation of catechol estrogens with a mixed mechanism of inhibition (competitive plus noncompetitive). Computational molecular modeling analysis showed that chlorogenic acid and caffeic acid can bind to human soluble COMT at the active site in a similar manner as the catechol estrogen substrates. Moreover, the binding energy values of these two coffee polyphenols are lower than that of catechol estrogens, which means that coffee polyphenols have higher binding affinity for the enzyme than the natural substrates. This computational finding agreed perfectly with our biochemical data.     

Hydroxamate-Stimulated O(2) Uptake in Roots of Pisum sativum and Zea mays, Mediated by a Peroxidase : Its Consequences for Respiration Measurements

Low concentrations of salicylhydroxamic acid (<5 millimolar) stimulate O₂ uptake in intact roots of Pisum sativum. We demonstrate that the hydroxamate-stimulated O₂ uptake does not reside in the mitochondria. We also show that the hydroxamate-stimulated O₂ uptake is due to the activation of a peroxidase catalyzing reduction of O₂. This peroxidase, which can use both NADH and NADPH as a substrate, is stimulated by low concentrations of monophenols, e.g. salicylhydroxamic acid and 2-methoxyphenol. It is inhibited by high (20 millimolar) concentrations of salicylhydroxamic acid, cyanide, and scavengers of the superoxide free radical ion, e.g. ascorbate, gentisic acid, and catechol. In the presence of gentisic acid, O₂ uptake by intact pea roots was no longer stimulated by low concentrations of salicylhydroxamic acid. The consequence of the present finding for in vivo respiration measurements is that the use of low concentrations of salicylhydroxamic acid and uncoupler is reliable only in the presence of a suitable superoxide free radical scavenger which prevents activation of the peroxidase. It also confirms that high concentrations of salicylhydroxamic acid (20-25 millimolar) can be safely used in short-term experiments to assess the activity of the alternative path in intact roots.     

Microbial oxidation of methanol: Properties of crystallized alcohol oxidase from a yeast, Pichia sp

Alcohol oxidase (alcohol:oxygen oxidoreductase) was crystallized from a methanolgrown yeast, Pichia sp. The crystalline enzyme is homogenous as judged from polyacrylamide gel electrophoresis. Alcohol oxidase catalyzed the oxidation of short-chain primary alcohols (C₁ to C₆), substituted primary alcohols (2-chloroethanol, 3-chloro-1-propanol, 4-chlorobutanol, isobutanol), and formaldehyde. The general reaction with an oxidizable substrate is as follows: Primary alcohol + O₂ → aldehyde + H₂O₂ Formaldehyde + O₂ → formate + H₂O₂. Secondary alcohols, tertiary alcohols, cyclic alcohols, aromatic alcohols, and aldehydes (except formaldehyde) were not oxidized. The K_m values for methanol and formaldehyde are 0.5 and 3.5 mm, respectively. The stoichiometry of substrate oxidized (alcohol or formaldehyde), oxygen consumed, and product formed (aldehyde or formate) is 1:1:1. The purified enzyme has a molecular weight of 300,000 as determined by gel filtration and a subunit size of 76,000 as determined by sodium dodecyl sulfate-gel electrophoresis, indicating that alcohol oxidase consists of four identical subunits. The purified alcohol oxidase has absorption maxima at 460 and 380 nm which were bleached by the addition of methanol. The prosthetic group of the enzyme was identified as a flavin adenine dinucleotide. Alcohol oxidase activity was inhibited by sulfhydryl reagents (p-chloromercuribenzoate, mercuric chloride, 5,5′-dithiobis-2-nitrobenzoate, iodoacetate) indicating the involvement of sulfhydryl groups(s) in the oxidation of alcohols by alcohol oxidase. Hydrogen peroxide (product of the reaction), 2-aminoethanol (substrate analogue), and cupric sulfate also inhibited alcohol oxidase activity.     

Purification and spectroscopic studies on catechol oxidases from Lycopus europaeus and Populus nigra: Evidence for a dinuclear copper center of type 3 and spectroscopic similarities to tyrosinase and hemocyanin

 We purified two catechol oxidases from Lycopus europaeus and Populus nigra which only catalyze the oxidation of catechols to quinones without hydroxylating tyrosine. The molecular mass of the Lycopus enzyme was determined to 39 800 Da and the mass of the Populus enzyme was determined to 56 050 Da. Both catechol oxidases are inhibited by thiourea, N-phenylthiourea, dithiocarbamate, and cyanide, but show different pH behavior using catechol as substrate. Atomic absorption spectroscopic analysis found 1.5 copper atoms per protein molecule. Using EPR spectroscopy we determined 1.8 Cu per molecule catechol oxidase. Furthermore, EPR spectroscopy demonstrated that catechol oxidase is a copper enzyme of type 3. The lack of an EPR signal is due to strong antiferromagnetic coupling that requires a bridging ligand between the two copper ions in the met preparation. Addition of H₂O₂ to both enzymes leads to oxy catechol oxidase. In the UV/Vis spectrum two new absorption bands occur at 345 nm and 580 nm. In accordance with the oxy forms of hemocyanin and tyrosinase the absorption band at 345 nm is due to an O₂ ^2– (π_σ*)→Cu(II) (d _x2–y2 ) charge transfer (CT) transition. The absorption band at 580 nm corresponds to the second O₂ ^2– (π_v*)→Cu(II) (d _x2–y2 ) CT transition. The UV/Vis bands in combination with the resonance Raman spectra of oxy catechol oxidase indicate a μ-η² : η² binding mode for dioxygen. The intense resonance Raman peak at 277 cm^–1, belonging to a Cu-N (axial His) stretching mode, suggests that catechol oxidase has six terminal His ligands, as known for molluscan and arthropodan hemocyanin. Received: 30 July 1998 / Accepted: 26 October 1998     

Catechol O-methyltransferases in pampas grass: differentiation of m- and p-methylating activities

Partially purified catechol O-methyltransferase from pampas grass (Cortaderia selloana) catalyses the methylations of substrates at both their meta and para positions. This capability was shown, by heat treatments, to arise from a less stable m-O-methyl-transferring activity and a more stable p-O-methyltransferring activity, tested against protocatechuic acid. When acting upon caffeic acid, the preparation catalyses a reaction of solely m-O-methyltransfer (in contrast to the mixed methylation of this substrate exhibited by rat liver catechol O-methyltransferase). A small degree of m-O-methylation of monophenolic substrates also occurs.     

High activity catechol 2,3-dioxygenase from the cresols - Degrading Stenotrophomonas maltophilia strain KB2

This study aimed at characterization of catechol 2,3-dioxygenase from Stenotrophomonas maltophilia KB2, being able to utilize a wide spectrum of aromatic substrates as a sole carbon and energy source. 2-methylphenol, 3-methylphenol, and 4-methylphenol was completely degraded during 24 h in concentration 6 mM, 7 mM, and 5 mM, respectively. When cells of strain KB2 were growing on methylphenols, catechol 2,3-dioxygenase was induced. Biochemical analysis revealed that the examined enzyme was similar to another catechol 2,3-dioxygenases, but showed extremely high activity. The enzyme was optimally active at 30 °C and pH 7.6. Kinetic studies showed that the value of K_m, V_max and Hill constant was 85.11 ??M, 3.08 ??M min⁻¹ and 4.09 respectively. Comparative structural and phylogenetic analysis of catechol 2,3-dioxygenase from S. maltophilia KB2 had placed the protein with the single-ring substrate subfamily of the extradiol dioxygenase. We observed the presence of externally located ??-helices and internally located ??-sheets. We also suggest that the Fe²⁺ ion binding is facilitated via four ligands: two histidine residues, one glutamate residue and one molecule of water.     

Oxidation and reduction of membrane-bound cytochrome c in Hemophilus parainfluenzae. Reaction with oxygen,hydrogen peroxide and nitrate

Cytochromes of the a-, b-, c- and d-type become reduced when intact cells of Hemophilus parainfluenzae have become anaerobic following respiration with substrates such as formate or succinate, as shown previously (J. Biol. Chem. (1970) 254, 5096–5100). In the presence of formate after depletion of O₂, there is an unusual two-step time course of reduction of the membrane-bound cytochrome c. The proportion of the cytochrome c which is reduced during the second stage is oxidizable by either nitrate or H₂O₂ and is reduced again when the nitrate or H₂O₂ have been depleted. We conclude that the observed two-stage reduction of cytochrome c results from the presence of an oxidant, probably H₂O₂, produced by reaction of formate dehydrogenase with O₂. This was shown by the effects of cyanide, catalase and O₂. In addition, no evidence for the production of the oxidant is seen when succinate is the substrate oxidized. Although measurements of absorption spectra indicated only one species of cytochrome c, kinetic evidence is presented for some separation of the cytochrome c into more than one electron transport pathway.     

High activity catechol 1,2-dioxygenase from Stenotrophomonas maltophilia strain KB2 as a useful tool in cis,cis-muconic acid production

This is the first report of a catechol 1,2-dioxygenase from Stenotrophomonas maltophilia strain KB2 with high activity against catechol and its methyl derivatives. This enzyme was maximally active at pH 8.0 and 40 °C and the half-life of the enzyme at this temperature was 3 h. Kinetic studies showed that the value of K _m and V _max was 12.8 μM and 1,218.8 U/mg of protein, respectively. During our studies on kinetic properties of the catechol 1,2-dioxygenase we observed substrate inhibition at >80 μM. The nucleotide sequence of the gene encoding the S. maltophilia strain KB2 catechol 1,2-dioxygenase has high identity with other catA genes from members of the genus Pseudomonas. The deduced 314-residue sequence of the enzyme corresponds to a protein of molecular mass 34.5 kDa. This enzyme was inhibited by competitive inhibitors (phenol derivatives) only by ca. 30 %. High tolerance against condition changes is desirable in industrial processes. Our data suggest that this enzyme could be of use as a tool in production of cis,cis-muconic acid and its derivatives.     

Photooxidation of ferrocyanide and iodide ions and associated phosphorylation in NH2OH-treated chloroplasts

NH₂OH-treated, non-water oxidizing chloroplasts are shown to be capable of oxidizing ferrocyanide and I^? via Photosystem II at appreciable rates (? 200 μequiv/h per mg chlorophyll). Using methylviologen as electron acceptor, ferrocyanide oxidation can be measured as O₂ uptake, as ferricyanide formation, or as H⁺ consumption (2 Fe²⁺ + 2H⁺ + O₂ → 2 Fe³⁺ + H₂O₂). I^? oxidation can be measured as methylviologen-mediated O₂ uptake, or spectrophotometrically, using ferricyanide as electron acceptor. The oxidation product I₂ is re-reduced, as it is formed, by unknown reducing substances in the reaction system.The rate-saturating concentrations of these donors are very high: 30 mM with ferricyanide and 15 mM with I^?. Relatively lipophilic Photosystem II donors such as catechol, benzidine and p-aminophenol saturate the photooxidation rate at much lower concentrations (< 0.5 mM). It thus seems that the oxidation of hydrophilic reductants such as ferricyanide and I^? is limited by permeability barriers. Very likely the site of Photosystem II oxidation is embedded in the thylakoid membrane or is situated on the inner surface of the membrane.The efficiency of phosphorylation (P/e₂) is 0.5 to 0.6 with ferrocyanide and about 0.5 with I^?. In contrast the P/e₂ ratio is 1.0 to 1.2 when water, catechol, p-aminophenol or benzidine serves as electron donor. These differences imply that only one of two phosphorylation sites operate when ferrocyanide and I^? are oxidized. Ferrocyanide and I^? are also chemically distinct from other Photosystem II donors in that their oxidation does not involve proton release. It is suggested that the mechanism of energy conservation associated with Photosystem II may be only operative when the removal of electrons from the donor results in release of protons (i.e. with water, hydroquinones, phenylamines, etc.).     

Catechol metabolites of the mycotoxin zearalenone are poor substrates but potent inhibitors of catechol-O-methyltransferase

The mycotoxin zearalenone (ZEN) elicits estrogenic effects and is biotransformed to two catechol metabolites, in analogy to the endogenous steroidal estrogen 17ß-estradiol (E2). Previous studies have shown that the catechol metabolites of ZEN have about the same potency to induce oxidative DNA damage as the catechol metabolites of E2, but are less efficiently converted to their methyl ethers by human hepatic catechol-O-methyltransferase (COMT). Here, we report that the two catechol metabolites of ZEN, i.e. 13-hydroxy-ZEN and 15-hydroxy-ZEN, are not only poor substrates of human COMT but are also able to strongly inhibit the O-methylation of 2-hydroxy-E2, the major catechol metabolite of E2. 15-Hydroxy-ZEN acts as a non-competitive inhibitor and is about ten times more potent than 13-hydroxy-ZEN, which is an uncompetitive inhibitor of COMT. The catechol metabolites of ZEN were also shown to inhibit the O-methylation of 2-hydroxy-E2 by hepatic COMT from mouse, rat, steer and piglet, although to a lesser extent than observed with human COMT. The powerful inhibitory effect of catechol metabolites of ZEN on COMT may have implications for the tumorigenic activity of E2, because catechol metabolites of E2 elicit genotoxic effects, and their impaired O-methylation may increase the tumorigenicity of steroidal estrogens.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor

NH₂OH-treated, non-water-splitting chloroplasts can oxidize H₂O₂ to O₂ through Photosystem II at substantial rates (100–250 μequiv · h^?1 · mg^?1 chlorophyll with 5 mM H₂O₂) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H₂O₂ → Photosystem II → dimethylquinone reaction supports phosphorylation with a $\text{P}\text{e}{}_2$ ratio of 0.25–0.35 and proton uptake with $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.67 (pH 8)–0.85 (pH 6). These are close to the $\text{P}\text{e}{}_2$ value of 0.3–0.38 and the $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.7–0.93 found in parallel experiments for the H₂O → Photosystem II → dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor → Photosystem II → dibromothymoquinone (→O₂) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I^?, ferrocyanide).     

Catalase evolved to concentrate H2O2 at its active site

Catalase is a homo-tetrameric enzyme that has its heme active site deeply buried inside the protein. Its only substrate, hydrogen peroxide (H₂O₂), reaches the heme through a 45 Å-long channel. Large-subunit catalases, but not small-subunit catalases, have a loop (gate loop) that interrupts the major channel. Two accesses lead to a gate that opens the final section of the channel to the heme; gates from the R-related subunits are interconnected. Using molecular dynamic simulations of the Neurospora crassa catalase-1 tetramer in a box of water (48,600 molecules) or 6 M H₂O₂, it is shown that the number of H₂O₂ molecules augments at the surface of the protein and in the accesses to the gate and the final section of the channel. Increase in H₂O₂ is due to the prevalence and distribution of amino acids that have an increased residency for H₂O₂ (mainly histidine, proline and charged residues), which are localized at the protein surface and the accesses to the gate. In the section of the channel from the heme to the gate, turnover rate of water molecules was faster than for H₂O₂ and increased residence sites for water and H₂O₂ were determined. In the presence of H₂O₂, the exclusion of water molecules from a specific site suggests a mechanism that could contend with the competing activity of water, allowing for catalase high kinetic efficiency.     

Experimentally calibrated computational chemistry of tryptophan hydroxylase: Trans influence, hydrogen-bonding, and 18-electron rule govern O2-activation

Insight into the nature of oxygen activation in tryptophan hydroxylase has been obtained from density functional computations. Conformations of O₂-bound intermediates have been studied with oxygen trans to glutamate and histidine, respectively. An O₂-adduct with O₂trans to histidine (O_his) and a peroxo intermediate with peroxide trans to glutamate (P_glu) were found to be consistent (0.57-0.59 mm/s) with experimental Mössbauer isomer shifts (0.55 mm/s) and had low computed free energies. The weaker trans influence of histidine is shown to give rise to a bent O₂ coordination mode with O₂ pointing towards the cofactor and a more activated O-O bond (1.33 Å) than in O_glu (1.30 Å). It is shown that the cofactor can hydrogen bond to O₂ and activate the O-O bond further (from 1.33 to 1.38 Å). The O_his intermediate leads to a ferryl intermediate (F_his) with an isomer shift of 0.34 mm/s, also consistent with the experimental value (0.25 mm/s) which we propose as the structure of the hydroxylating intermediate, with the tryptophan substrate well located for further reaction 3.5 Å from the ferryl group. Based on the optimized transition states, the activation barriers for the two paths (glu and his) are similar, so a two-state scenario involving O_his and P_glu is possible. A structure of the activated deoxy state which is high-spin implies that the valence electron count has been lowered from 18 to 16 (glutamate becomes bidentate), giving a “green light” that invites O₂-binding. Our mechanism of oxygen activation in tryptophan hydroxylase does not require inversion of spin, which may be an important observation.     

Studies on the Energy-coupling Sites of Photophosphorylation: V. Phosphorylation Efficiencies (P/e(2)) Associated with Aerobic Photooxidation of Artificial Electron Donors

The rate of Hill reaction can be measured accurately as O₂ uptake (the Mehler reaction) if a rapidly autoxidizable electron acceptor (e.g., methylviologen) is used. However, when an artificial electron donor-ascorbate couple (or ascorbate alone) replaces the natural donor, water, the rate of O₂ consumption is no longer a reliable measure of the electron flux, because superoxide radical reactions contribute to O₂ uptake. Such radical reactions, however, can be suppressed by adding enough superoxide dismutase to the reaction mixture. Indeed in all of the photosystem I- and photosystem II-donor reactions tested (except with benzidine which was tested without ascorbate added), the O₂ uptake was inhibited by 30 to 50% by the addition of superoxide dismutase. The rate of phosphorylation was totally unaffected by the enzyme. The reasessment of the phosphorylation efficiencies thus made by the use of superoxide dismutase led us to the following conclusions. The phosphorylation efficiency associated with the transfer of electrons from a donor to methlylviologen (than to O₂) through both photosystems II and I is practically independent of the donor used—catechol, benzidine, p-aminophenol, dicyanohydroquinone, or water. The P/e₂ ratio is 1.0 ± 0.1. Only ascorbate gives a slightly lower value (P/e₂ = 0.9). (NH₂OH-treated, non-water-splitting chloroplasts were used for reactions with these artificial donors.) The phosphorylation efficiency associated with DCMU-insensitive, photosystem I-mediated transfer of electrons from a donor to methylviologen (then to O₂) is again largely independent of the donor used, such as diaminodurene, diaminotoluene, and reduced 2,6-dichlorphenol-indophenol. The P/e₂ ratio is 0.6 ± 0.08.     

Developmental changes in the responses of O2 uptake and ventilation to acutely declining O2 tensions in larval krill Meganyctiphanes norvegica

The effect of exposure to acutely declining oxygen tensions on O₂ uptake (M_O2) and ventilation has been investigated in different larval stages of Northern krill Meganyctiphanes norvegica (calytopis III/early furcilia I, late furcilia I, furcilia III and V). An ability to regulate M_O2 during acutely declining P_O2 began to appear about furcilia III (critical O₂ tension or P_c=15.4±0.73 kPa) and had improved by furcilia V (P_c=12.6±0.39 kPa). Hypoxia-related hyperventilation was achieved by an increase in pleopod (but not thoracic limb) activity (P_c∼11 kPa), a sensitivity which also appeared at, or just before, furcilia V even though an earlier stage (furcilia III) had a full compliment of functional setose pleopods. While this regulatory ability appeared as the gills were beginning to form, furcilia V is still early in gill ontogeny compared with adults. Preexposure to very moderate hypoxia (60% and 70% O₂ saturation) of furcilia III and V resulted in substantial mortality, but where it did not (furcilia V, 80% O₂ saturation), there was no effect of keeping krill at this P_O2 on either M_O2 or ventilation, suggesting that the development of respiratory regulation in M. norvegica is not open to environmental influence in the same way as for other crustaceans. We suggest that ontogeny of pleopod control provides furcilia V+ with both a stronger means of propulsion, allowing the ontogeny of DVM but also with an ability to regulate M_O2 during exposure to acutely declining P_O2s. The onset of respiratory regulation (furcilia V) preceded the onset of DVM (furcilia VI+). As pleopod ontogeny is associated intimately with the ontogeny of DVM and respiratory regulation, in the Gullmarsfjord this co-occurrence is fortuitous as krill can be required during DVM to migrate into hypoxic water which they are not equipped to deal with, in physiological terms, before furcilia V.