Children with psoriasis and COVID-19: factors associated with an unfavourable COVID-19 course, and the impact of infection on disease progression (Chi-PsoCov registry).

BACKGROUND: The COVID-19 pandemic has raised questions regarding the management of chronic skin diseases, especially in patients on systemic treatments. Data concerning the use of biologics in adults with psoriasis are reassuring, but data specific to children are missing. Moreover, COVID-19 could impact the course of psoriasis in children. OBJECTIVES: The aim of this study was therefore to assess the impact of COVID-19 on the psoriasis of children, and the severity of the infection in relation to systemic treatments. METHODS: We set up an international registry of paediatric psoriasis patients. Children were included if they were under 18 years of age, had a history of psoriasis, or developed it within 1 month of COVID-19 and had COVID-19 with or without symptoms. RESULTS: One hundred and twenty episodes of COVID-19 in 117 children (mean age: 12.4 years) were reported. The main clinical form of psoriasis was plaque type (69.4%). Most children were without systemic treatment (54.2%); 33 (28.3%) were on biologic therapies, and 24 (20%) on non-biologic systemic drugs. COVID-19 was confirmed in 106 children (88.3%) and 3 children had two COVID-19 infections each. COVID-19 was symptomatic for 75 children (62.5%) with a mean duration of 6.5 days, significantly longer for children on non-biologic systemic treatments (P = 0.02) and without systemic treatment (P = 0.006) when compared with children on biologics. The six children who required hospitalization were more frequently under non-biologic systemic treatment when compared with the other children (P = 0.01), and particularly under methotrexate (P = 0.03). After COVID-19, the psoriasis worsened in 17 cases (15.2%). Nine children (8%) developed a psoriasis in the month following COVID-19, mainly a guttate form (P = 0.01). DISCUSSION: Biologics appear to be safe with no increased risk of severe form of COVID-19 in children with psoriasis. COVID-19 was responsible for the development of psoriasis or the worsening of a known psoriasis for some children.

SYMPTOMATOLOGY AND TREATMENT OF COVID-19 AFFECTING SKIN APPENDAGES: A NARRATIVE REVIEW BEYOND COVID-TOES.

SARS-CoV-2 is the cause of COVID-19 disease and responsible for a pandemic since the 2020. Multiple organ involvement has been described including cutaneous symptoms. Affection of skin appendages, however, seems to be under-reported except for COVID-toes. We performed a PUBMED research for "COVID-19" OR "SARS-CoV-2" AND "skin appendages", "hair", "nails", and "skin glands" from January 2020 to April 2022. COVID toes were excluded since this symptom had extensively been discussed. The focus of this narrative review was laid on clinical presentation, association to the course of COVID-19 disease and treatment options. Skin appendages can be affected by COVID-19 disease beyond COVID-toes, both by symptomatic and asymptomatic course. Telogen effluvium, androgenetic alopecia, and alopecia areata are the most common hair disorders in COVID-19 patients. Nails are less commonly affected by COVID-19 than hair. Splinter hemorrhages and leukonychia are the most frequent findings. While sebaceous glands seem to be uninvolved, SARS-CoV-2 spike proteins have been identified in eccrine sweat glands. Alopecia areata is often seen among asymptomatic COVID-19 patients while telogen effluvium is observed in symptomatic and asymptomatic patients. The half-moon sign on the nails could be a red flag for a more severe course of COVID-19. Treatment options are summarized. Skin appendages are not spared by COVID-19. Their knowledge will help to identify asymptomatic patients and patients at risk for a more severe course of the viral disease.

The abducens nerve: its topography and anatomical variations in intracranial course with clinical commentary.

BACKGROUND: The sixth cranial nerve (CN VI) - or the abducens nerve - in humans supplies only the lateral rectus muscle. Due to its topographic conditions, including angulations and fixation points along its course from the brainstem to the lateral rectus muscle, the CN VI is vulnerable to injury. Every case of CN VI palsy requires precise diagnostics, which is facilitated by an understanding of the anatomy. The present article's aims include a detailed study of the intracranial course of the CN VI, determination of occurrence of its particular anatomical variations, as well as presentation of some essential anatomical conditions which may conduce to CN VI palsy. Special emphasis was put on the correlation between craniometric measurements and a particular variation of the CN VI, which complements the data that can be found in literature. MATERIALS AND METHODS: Twenty randomly selected specimens of cadaveric heads fixed in a 10% formalin solution were studied. The study used 40 specimens of the CN VI in order to examine its course variations within the section between the pontomedullary sulcus and the superior orbital fissure. RESULTS: Detailed analysis of the CN VI topography and anatomy in its intracranial course revealed 3 anatomical variations of the nerve in the studied specimens. Variation I, found in 70% of cases, covers those cases in which the CN VI was found to be a single trunk. Those cases in which there was a branching of the CN VI exclusively inside the cavernous sinus were classified as variation II, occurring in 20% of cases. Cases of duplication of the CN VI were classified as variation III, found in 10% of the specimens. In 75% of cases of CN VI duplication one of the nerve trunks ran upwards from the petrosphenoidal ligament, outside Dorello's canal. CONCLUSIONS: The CN VI throughout its intracranial course usually runs as a single trunk, however, common variations include also branching of the nerve in the cavernous sinus or duplication. Topographic relations of the CN VI with adjacent structures account for the risk of injuries which may be caused to the nerve as a result of a disease or surgical procedures.

Partial Ribosomal Nontranscribed Spacer Sequences Distinguish Rhagoletis zephyria (Diptera: Tephritidae) From the Apple Maggot, R. pomonella.

The apple maggot, Rhagoletis pomonella (Walsh), was introduced into the apple-growing regions of the Pacific Northwest in the U.S.A. during the past 60-100 yr. Apple maggot (larvae, puparia, and adults) is difficult to distinguish from its morphologically similar sister species, Rhagoletis zephyria Snow, which is native and abundant in the Pacific Northwest. While morphological identifications are common practice, a simple, inexpensive assay based on genetic differences would be very useful when morphological traits are unclear. Here we report nucleotide substitution and insertion-deletion mutations in the nontranscribed spacer (NTS) of the ribosomal RNA gene cistron of R. pomonella and R. zephyria that appear to be diagnostic for these two fly species. Insertion-deletion variation is substantial and results in a 49 base-pair difference in PCR amplicon size between R. zephyria and R. pomonella that can be scored using agarose gel electrophoresis. PCR amplification and DNA sequencing of 766 bp of the NTS region from 38 R. pomonella individuals and 35 R. zephyria individuals from across their geographic ranges led to the expected PCR fragments of approx. 840 bp and 790 bp, respectively, as did amplification and sequencing of a smaller set of 26 R. pomonella and 16 R. zephyria flies from a sympatric site in Washington State. Conversely, 633 bp mitochondrial COI barcode sequences from this set of flies were polyphyletic with respect to R. pomonella and R. zephyria. Thus, differences in NTS PCR products on agarose gels potentially provide a simple way to distinguish between R. pomonella and R. zephyria.

Light-induced structural changes in cytochrome c oxidase: implication for the mechanism of electron and proton gating.

We have investigated electrogenic events and absorbance changes following pulsed illumination of partly reduced cytochrome c oxidase in the absence of dioxygen and carbon monoxide (Hallén et al. (1993) FEBS Lett. 318, 134-138). In both types of experiment similar kinetics were observed; a rapid (tau < 0.5 micros) change was followed by relaxations with time constants of approx. 7 micros and 80 micros. Both the time constant and the activation energy of the 80 micros component were, within the experimental error, the same as those of one of the steps in the reduction of dioxygen by reduced cytochrome c oxidase. The absorbance changes showed a rapid haem reduction, followed by reoxidation. They were affected by CN(-) and N(-)3, ligands which bind in the binuclear centre of cytochrome c oxidase; the absorbance changes were quenched by CN(-) and in the presence of N(-)3, the amplitude of the 7 micros component increased whereas that of the 80 micros decreased. Based on these findings, a model is proposed which involves electron transfer from Cu(+)B to Fe(3+)A3, as a response to structural changes upon pulsed illumination. The same structural changes are also suggested to take place in the oxygen reduction. These changes may play an important role in the gating of electrons as well as protons, an obligatory feature of a redox-linked proton pump.

The mechanism of electron gating in proton pumping cytochrome c oxidase: the effect of pH and temperature on internal electron transfer.

Electron-transfer reactions following flash photolysis of the mixed-valence cytochrome oxidase-CO complex have been measured at 445, 598 and 830 nm between pH 5.2 and 9.0 in the temperature range of 0-25 degrees C. There is a rapid electron transfer from the cytochrome a3-CuB pair to CuA (time constant: 14200 s-1), which is followed by a slower electron transfer to cytochrome a. Both the rate and the amplitude of the rapid phase are independent of pH, and the rate in the direction from CuA to cytochrome a3-CuB is practically independent of temperature. The second phase depends strongly on pH due to the titration of an acid-base group with pKa = 7.6. The equilibrium at pH 7.4 corresponds to reduction potentials of 225 and 345 mV for cytochrome a and a3, respectively, from which it is concluded that the enzyme is in a different conformation compared to the fully oxidized form. The results have been used to suggest a series of reaction steps in a cycle of the oxidase as a proton pump. Application of the electron-transfer theory to the temperature-dependence data suggests a mechanism for electron gating in the pump. Reduction of both cytochrome a and CuA leads to a conformational change, which changes the structure of cytochrome a3-CuB in such a way that the reorganizational barrier for electron transfer is removed and the driving force is increased.

Factors determining electron-transfer rates in cytochrome c oxidase: investigation of the oxygen reaction in the R. sphaeroides enzyme.

We have investigated the kinetics of the single-turnover reaction of fully reduced solubilised cytochrome c oxidase (cytochrome aa3) from Rhodobacter sphaeroides with dioxygen using the flow-flash methodology and compared the results to those obtained with the well-characterised bovine mitochondrial enzyme. The overall reaction sequence was the same in the two enzymes, but the extents and rates of the electron-transfer reactions differed, implying differences in redox potentials, and/or interaction energies between electrons and protons during oxygen reduction. As with the bovine enzyme, the R. sphaeroides enzyme displayed two major kinetic phases of proton uptake with rate constants of approximately 5000 s-1 and approximately 500 s-1 at pH 7.9, concomitant with the peroxy to oxoferryl and oxoferryl to oxidised states. The net number of protons taken up in the R. sphaeroides enzyme was about approximately 1.9, which implies that upon reduction, the enzyme has to pick up approximately 2.1 H+ from the medium. On the basis of the comparison of electron-transfer reactions in the two enzymes, we conclude that the transfer rate of the fourth electron to the binuclear centre is not only determined by the electron-transfer rate from haem a to the binuclear centre, but also by the electron equilibrium between CuA and haem a. In addition, in contrast to the bovine enzyme, where the electron- and proton-transfer rates during oxidation of the fully reduced enzyme by O2 are all faster than the overall turnover rate, in the R. sphaeroides enzyme, the slowest kinetic phase was rate limiting for the overall turnover. Moreover, the comparison of the reactions in the two systems shows that in the R. sphaeroides enzyme, the electrons are more evenly distributed among the redox centres during oxygen reduction. This enables investigations of effects also of minor perturbations on, e.g., the electron-transfer characteristics in mutant enzymes, for which this study forms the basis.

Photoinduced electron transfer from carboxymethylated cytochrome c to plastocyanin.

Photoinduced electron transfer from cytochrome c to plastocyanin was investigated using a novel method. Reduced carboxymethylated cytochrome c (CmCyt c), with carbon monoxide bound to the heme iron, and oxidized plastocyanin were mixed. At 1 mM CO the reduced state of CmCyt c is stabilized by about 350 meV. After flash photolysis of CO the apparent redox potential of CmCyt c drops resulting in electron transfer to plastocyanin. The electron transfer characteristics were investigated at approximately 30 different wavelengths in the range 390-460 nm. A global fit of the data showed that the electron transfer rate is 960+/-30 s-1 at pH 7.

The effect of pH and temperature on the reaction of fully reduced and mixed-valence cytochrome c oxidase with dioxygen.

The reaction of fully reduced and mixed-valence cytochrome oxidase with O2 has been followed in flow-flash experiments, starting from the CO complexes, at 428, 445, 605 and 830 nm between pH 5.8b and 9.0 in the temperature range of 2-40 degrees C. With the fully reduced enzyme, four kinetic phase with rate constants at pH 7.4 and 25 degrees C of 9 x 10(4), 2.5 x 10(4), 1.0 x 10(4) and 800 s(-1), respectively, are observed. The rates of the three last phases display a very small temperature dependence, corresponding to activation energies in the range 13-54 kJ x mol(-1). The rates of the third and fourth phases decrease at high pH due to the deprotonation of groups with pKa values of 8.3 and 8.8, respectively, but also the second phase appears to have a small pH dependence. In the reaction of the mixed-valence enzyme, three kinetic phases with rate constants at pH 7.4 and 25 degrees C of 9 x 10(4), 6000 and 150 s(-1), respectively, are observed. The third phase only has a small temperature dependence, corresponding to an activation energy of 20 kJ x mol(-1). No pH dependence could be detected for any phase. Reaction schemes consistent with the experimental observations are presented. The pH dependencies of the rates of the two final phase in the reaction of the fully reduced enzyme are proposed to be related to the involvement of protons in the reduction of a peroxide intermediate. The temperature dependence data suggest that the reorganization energies and driving forces are closely matched in all electron transfer steps with both enzyme forms. It is suggested that the slowest step in the reaction of the mixed-valence enzyme is a conformation change involved in the reaction cycle of cytochrome oxidase as a proton pump.

The catalytic mechanism of gastric H+/K+-ATPase: simulations of pre-steady-state and steady-state kinetic results.

A reaction cycle for the gastric H+/K+-ATPase is proposed. This has been used to simulate the results from four types of pre-steady-state and steady-state kinetic experiments: (1) the K+ dependence of the dephosphorylation of the phosphoenzyme; (2) the rate of phosphorylation of the enzyme by ATP at different concentrations; (3) the effect of ATP concentration on the steady-state rate of ATP hydrolysis; (4) the phosphoenzyme levels in the steady state at various ATP concentrations. A single set of equilibrium and rate constants can be used to reproduce the results from all four sets of experiments quite well. It is suggested that the steady-state rate equation is nonhyperbolic because ATP can react with the enzyme in both the E1 and the E2 state, but with a lower affinity in E2. No single step is by itself limiting the maximum turnover rate.

Light-induced electrogenic events associated with proton uptake upon forming QB- in bacterial wild-type and mutant reaction centers.

Light-induced voltage changes (electrogenic events) were measured in wild-type and site-directed mutants of reaction centers (RCs) from Rhodobacter sphaeroides oriented in a lipid monolayer adsorbed to a Teflon film. A rapid increase in voltage associated with charge separation was followed by a slower increase attributed to proton transfer from solution to protonatable amino-acid residues in the vicinity of the QB site. In native reaction centers the proton-transfer voltage had a pH-dependent amplitude with two peaks at pH 4.5 and pH 9.7, respectively. In the Glu-L212-->Gln RCs the high-pH peak was absent, whereas in the Asp-L213-->Asn RCs the low-pH peak was absent and the high-pH peak was shifted to lower pH by about 1.3 pH units. The amplitudes of the electrogenic phases as a function of pH follow approximately the measured proton uptake from solution (P.H. McPherson, M.Y. Okamura, G. Feher, Biochim. Biophys. Acta, vol. 934, 1988, pp. 348-368) and are ascribed to proton transfer to amino acid residues upon QB- formation. The peak around pH 9.7 is ascribed to proton uptake predominantly by Glu-L212 and the peak around pH 4.5 to proton uptake predominantly by Asp-L213 or a residue strongly interacting with Asp-L213.

Proton transfer from glutamate 286 determines the transition rates between oxygen intermediates in cytochrome c oxidase.

We have investigated the electron-proton coupling during the peroxy (P(R)) to oxo-ferryl (F) and F to oxidised (O) transitions in cytochrome c oxidase from Rhodobacter sphaeroides. The kinetics of these reactions were investigated in two different mutant enzymes: (1) ED(I-286), in which one of the key residues in the D-pathway, E(I-286), was replaced by an aspartate which has a shorter side chain than that of the glutamate and, (2) ML(II-263), in which the redox potential of Cu(A) is increased by approximately 100 mV, which slows electron transfer to the binuclear centre during the F-->O transition by a factor of approximately 200. In ED(I-286) proton uptake during P(R)-->F was slowed by a factor of approximately 5, which indicates that E(I-286) is the proton donor to P(R). In addition, in the mutant enzyme the F-->O transition rate displayed a deuterium isotope effect of approximately 2.5 as compared with approximately 7 in the wild-type enzyme. Since the entire deuterium isotope effect was shown to be associated with a single proton-transfer reaction in which the proton donor and acceptor must approach each other (M. Karpefors, P. Adelroth, P. Brzezinski, Biochemistry 39 (2000) 6850), the smaller deuterium isotope effect in ED(I-286) indicates that proton transfer from E(I-286) determines the rate also of the F-->O transition. In ML(II-263) the electron-transfer to the binuclear centre is slower than the intrinsic proton-transfer rate through the D-pathway. Nevertheless, both electron and proton transfer to the binuclear centre displayed a deuterium isotope effect of approximately 8, i.e., about the same as in the wild-type enzyme, which shows that these reactions are intimately coupled.

Kinetics of electron and proton transfer during O(2) reduction in cytochrome aa(3) from A. ambivalens: an enzyme lacking Glu(I-286).

Acidianus ambivalens is a hyperthermoacidophilic archaeon which grows optimally at approximately 80 degrees C and pH 2.5. The terminal oxidase of its respiratory system is a membrane-bound quinol oxidase (cytochrome aa(3)) which belongs to the heme-copper oxidase superfamily. One difference between this quinol oxidase and a majority of the other members of this family is that it lacks the highly-conserved glutamate (Glu(I-286), E. coli ubiquinol oxidase numbering) which has been shown to play a central role in controlling the proton transfer during reaction of reduced oxidases with oxygen. In this study we have investigated the dynamics of the reaction of the reduced A. ambivalens quinol oxidase with O(2). With the purified enzyme, two kinetic phases were observed with rate constants of 1.8&z.ccirf;10(4) s(-1) (at 1 mM O(2), pH 7.8) and 3. 7x10(3) s(-1), respectively. The first phase is attributed to binding of O(2) to heme a(3) and oxidation of both hemes forming the 'peroxy' intermediate. The second phase was associated with proton uptake from solution and it is attributed to formation of the 'oxo-ferryl' state, the final state in the absence of quinol. In the presence of bound caldariella quinol (QH(2)), heme a was re-reduced by QH(2) with a rate of 670 s(-1), followed by transfer of the fourth electron to the binuclear center with a rate of 50 s(-1). Thus, the results indicate that the quinol donates electrons to heme a, followed by intramolecular transfer to the binuclear center. Moreover, the overall electron and proton-transfer kinetics in the A. ambivalens quinol oxidase are the same as those in the E. coli ubiquinol oxidase, which indicates that in the A. ambivalens enzyme a different pathway is used for proton transfer to the binuclear center and/or other protonatable groups in an equivalent pathway are involved. Potential candidates in that pathway are two glutamates at positions (I-80) and (I-83) in the A. ambivalens enzyme (corresponding to Met(I-116) and Val(I-119), respectively, in E. coli cytochrome bo(3)).

Electron-proton interactions in terminal oxidases.

The cytochrome c and ubiquinol oxidases discussed in this article are membrane-bound redox-driven proton pumps which couple an electron current to a proton current across the membrane. This coupling requires a control of the thermodynamics and/or rates of internal electron- and proton-transfer reactions (termed 'gating'). Therefore, to understand the structure-function relation of these proton pumps, individual electron- and proton-transfer reactions must be investigated. We have undertaken such studies by using a combination of site-directed mutagenesis and spectroscopic techniques. The results show that proton uptake/release upon reduction/oxidation of heme a3 takes place on a ms-time scale through the K-pathway (including Thr(I-359) and Lys(I-362)), but not through the D-pathway (including Asp(I-132) and Glu(I-286)). During reaction of the reduced enzyme with O2, both substrate and pumped protons are taken up through the D-pathway (but not through the K-pathway) in a biphasic process with time constants of 100 microseconds and 1 ms. Thus, the original assignment of the role of the D-pathway (used only for pumped protons) must be revised. Dynamic studies of proton uptake to the enzyme surface show that on the proton-input side, the surface carries a proton-collecting antenna made of carboxylate and histidine residues which enable the enzyme to pick up protons with a rate compatible to the enzyme turnover rate. These results are consistent with the three-dimensional cytochrome c oxidase structure which shows that the entry point to the D-pathway (but not to the K-pathway) is surrounded by a network of histidine residues within a negative electrostatic potential.

Zinc ions inhibit oxidation of cytochrome c oxidase by oxygen.

Cytochrome c oxidase is a membrane-bound enzyme that catalyses the reduction of O2 to H2O and uses part of the energy released in this reaction to pump protons across the membrane. We have investigated the effect of addition of Zn2+ on the kinetics of two reaction steps in cytochrome c oxidase that are associated with proton pumping; the peroxy to oxo-ferryl (P(r)-->F) and the oxo-ferryl to oxidised (F-->O) transitions. The Zn2+ binding resulted in a decrease of the F-->O rate from 820 s(-1) (no Zn2+) to a saturating value of approximately 360 s(-1) with an apparent K(D) of approximately 2.6 microM. The P(r)-->F rate (approximately 10[(4) s(-1)] before addition of Zn2+) decreased more slowly with increasing Zn2+ concentration and a K(D) of approximately 120 microM was observed. The effects on both kinetic phases were fully reversible upon addition of EDTA. Since both the P(r)-->F and F-->O transitions are associated with proton uptake through the D-pathway, a Zn2+-binding site is likely to be located at the entry point of this pathway, where several carboxylates and histidine residues are found that may co-ordinate Zn2+.

Light-induced structural changes in cytochrome c oxidase. Measurements of electrogenic events and absorbance changes.

We have investigated flash-induced electrogenic events and absorbance changes in cytochrome c oxidase in the absence of dioxygen and carbon monoxide. Electrogenic events were studied using a Teflon-bound layer of cytochrome c oxidase oriented in a phospholipid monolayer. Absorbance changes were observed exclusively in partly reduced cytochrome c oxidase; the largest changes were found in the one-electron-reduced species. Electrogenic events were detected in all reduction states of the enzyme. Both types of experiments displayed a rapid (< 0.5 microseconds) event followed by a biphasic relaxation. The time constants of the relaxation were 6 +/- 2 microseconds and 70 +/- 10 microseconds in the electrogenicity, and 9 +/- 3 microseconds in the absorbance changes (at approximately 22 degrees C). The kinetic absorbance difference spectrum was consistent with that of reduced minus oxidized haem. The experimental results are discussed in terms of structural changes in the vicinity of cytochrome a3. These changes may play an important role in all studies that involve flash photolysis of cytochrome c oxidase-ligand complexes.

The reduction of cytochrome c oxidase by carbon monoxide.

The rate of formation of the mixed-valence state of cytochrome c oxidase on incubation with carbon monoxide is strongly dependent on pH, supporting the concept that CO itself is the reducing agent. The reaction is biphasic due to the presence of two different enzyme forms in the resting oxidase. The kinetics of the reaction with the major enzyme form suggests an initial rapid binding of CO in a heme pocket of the oxidized enzyme and a subsequent slow intramolecular electron transfer to the cytochrome alpha 3-CuB site. Cytochrome alpha and CuA are not reduced even on prolonged incubation.

The rate-limiting step and nonhyperbolic kinetics in the oxidation of ferrocytochrome c catalyzed by cytochrome c oxidase.

The level of reduction of cytochrome a and CuA during the oxidation of ferrocytochrome c has been determined in stopped-flow experiments. Both components are partially reduced but become progressively more oxidized as the reaction proceeds. When all cytochrome c has been oxidized, CuA is also completely oxidized, whereas cytochrome a is still partially reduced. These results can be simulated on the basis of a model which requires that the intramolecular electron transfer from cytochrome a and CuA to cytochrome a3-CuB is a two-electron process and, in addition, that the binding of oxidized cytochrome c to the electron- transfer site decreases the rate constants for intramolecular electron transfer from cytochrome a. The first requirement is related to the function of the oxidase as a proton pump. Product dissociation is not by itself rate-limiting, making it less likely that the source of the nonhyperbolic substrate kinetics is an effect on this step from electrostatic interaction with ferricytochrome c bound to a second site. It is pointed out that nonhyperbolic kinetics is, in fact, an intrinsic property of ion pumps.

Two-electron reduction is required for rapid internal electron transfer in resting, pulsed and oxygenated cytochrome c oxidase.

The resting as well as the 420 nm and 428 nm forms of cytochrome oxidase have been studied in kinetic experiments with an excess of enzyme over reduced cytochrome c. No difference was found in the behavior of the two activated forms. With all three forms, a fraction of cytochrome a was reoxidized with a rate which was much lower than kcat. This suggests that intramolecular transfer to the dioxygen-reducing site occurs only if both cytochrome a and CuA are reduced. An initial rapid phase in the oxidation of cytochrome a in the pulsed and oxygenated enzymes is related to the presence of a three-electron-reduced dioxygen intermediate. The increased catalytic activity of pulsed and oxygenated oxidase can be explained on the basis of a shift in the redox equilibrium between cytochrome a and CuA.