Chemotaxis by Pseudomonas putida (ATCC 17453) towards camphor involves cytochrome P450cam (CYP101A1)

The camphor-degrading microorganism, Pseudomonas putida strain ATCC 17453, is an aerobic, gram-negative soil bacterium that uses camphor as its sole carbon and energy source. The genes responsible for the catabolic degradation of camphor are encoded on the extra-chromosomal CAM plasmid. A monooxygenase, cytochrome P450_cam, mediates hydroxylation of camphor to 5-exo-hydroxycamphor as the first and committed step in the camphor degradation pathway, requiring a dioxygen molecule (O₂) from air. Under low O₂ levels, P450_cam catalyzes the production of borneol via an unusual reduction reaction. We have previously shown that borneol downregulates the expression of P450_cam. To understand the function of P450_cam and the consequences of down-regulation by borneol under low O₂ conditions, we have studied chemotaxis of camphor induced and non-induced P. putida strain ATCC 17453. We have tested camphor, borneol, oxidized camphor metabolites and known bacterial attractants (d)-glucose, (d) - and (l)-glutamic acid for their elicitation chemotactic behavior. In addition, we have used 1-phenylimidazole, a P450_cam inhibitor, to investigate if P450_cam plays a role in the chemotactic ability of P. putida in the presence of camphor. We found that camphor, a chemoattractant, became toxic and chemorepellent when P450_cam was inhibited. We have also evaluated the effect of borneol on chemotaxis and found that the bacteria chemotaxed away from camphor in the presence of borneol. This is the first report of the chemotactic behaviour of P. putida ATCC 17453 and the essential role of P450_cam in this process.     

A complete volume profile for the reversible binding of camphor to cytochrome P450<Subscript>cam</Subscript>

The effect of pressure on the kinetics and thermodynamics of the reversible binding of camphor to cytochrome P450_cam was studied as a function of the K⁺ concentration. The determination of the reaction and activation volumes enabled the construction of the first complete volume profile for the reversible binding of camphor to P450_cam. Although the volume profiles constructed for the reactions conducted at low and high K⁺ concentrations are rather similar, and both show a drastic volume increase on going from the reactant to the transition state and a relatively small volume change on going from the transition to the product state, the position of the transition state is largely affected by the K⁺ concentration in solution. Similarly, the activation volume determined for the dissociation of camphor is influenced by the presence of K⁺, which reflects changes in the ease of water entering the active site of camphor-bound P450_cam that depends on the K⁺ concentration. Careful analysis of the components that contribute to the observed volume changes allowed the estimation of the total number of water molecules expelled to the bulk solvent during the binding of camphor to P450_cam and the subsequent spin transition. The results are discussed in reference to other studies reported in the literature that deal with the kinetics and thermodynamics of the binding of camphor to P450_cam under various reaction conditions.     

Linking of cytochrome P450cam and putidaredoxin by a co-ordination bridge

Cytochrome P450_cam (CYP101) catalyzes the oxidation of D(+)-camphor at the 5 position. The enzyme couples the reduction of dioxygen to the oxidation of the substrate. To transfer electrons from the reductant (NADH) to the cytochrome, two additional proteins are required. These are putidaredoxin (PdX) and putidaredoxin reductase (PdR). We have chemically linked a form of PdX with a histidine tag at the C-terminus to the P450. To accomplish this, we have modified cysteine 334 on P450 with a bipyridinyl group, and co-ordinated the C-terminal histidine tag of PdX by the addition of Ni²⁺ or Ru³⁺. The Ru³⁺ complex was the most stable. The non-linked system gave mostly 5-ketocamphor, a product of two consecutive hydroxylations, and H₂O₂, a product of 2-electron uncoupling. The Ni²⁺ complex gave both 5-exo-hydroxycamphor and 5-ketocamphor, but it also uncoupled. The Ru³⁺ complex gave a single product (5-exo-hydroxycamphor) and did not uncouple at the optimal PdR concentration. Our results are consistent with other studies of this system, in that strong binding of PdX to P450 is crucial for good coupling and for release of 5-exo-hydroxycamphor.     

Efficient catalytic turnover of cytochrome P450(cam) is supported by a T252N mutation

A Thr (or Ser) residue on the I-helix is a highly conserved structural feature of cytochrome P450 enzymes. It is believed to be indispensable as a proton delivery shuttle in the oxygen activation process. Previous work showed that P450_cin (CYP176A1), which contains an Asn instead of the conserved Thr, is fully functional in the catalytic oxidation of cineole [D.B. Hawkes, G.W. Adams, A.L. Burlingame, P.R. Ortiz de Montellano, J.J. De Voss, J. Biol. Chem. 277 (2002) 27725-27732]. To determine whether the substitution of Asn for Thr is specific or general, the conserved Thr252 in P450_cam (CYP101) was mutated to generate the T252N, T252N/V253T, and T252A mutants. Steady-state kinetic analysis of the oxidation of camphor by these mutants indicated that the T252N and T252N/V253T mutants have comparable turnover numbers but higher K_m values relative to the wild-type enzyme. Spectroscopic binding assays indicate that the higher K_m values reflect a decrease in the camphor binding affinity. Non-productive H₂O₂ generation was negligible with the T252N and T252N/V253T mutants, but, as previously observed, was dominant in the T252A mutant. Our results, and a structure model based on the crystal structures of the ferrous dioxygen complexes of P450_cam and its T252A mutant, suggest that Asn252 can stabilize the ferric hydroperoxy intermediate, preventing premature release of H₂O₂ and enabling addition of the second proton to the distal oxygen to generate the catalytic ferryl species.     

Caffeine-inducible enzyme activity in Pseudomonas putida ATCC 700097

Caffeine (1,3,7-trimethylxanthine), a ubiquitous component of human diet has been suggested as a chemical indicator of ecosystem impacts of sewage spills and treated effluent discharges because it is not sufficiently metabolized by wastewater microorganisms. This study identified enzymes responsible for caffeine metabolism in sewage bacteria. Pseudomonas putida biotype A (ATCC 700097) originally isolated as a rare caffeine-degrading organism in domestic wastewater exhibited diauxic growth on caffeine, concomitant with the expression of a P450-type cytochrome and peroxidase enzyme activities. Initial growth phase lasted 13.8 ± 1.4 h with a growth rate that was five times slower than the secondary growth phase that lasted 5.5 ± 1.2 h. Molecular and enzymatic characteristics of the cytochrome P450-type enzyme differ from the previously described cytochrome P450 (P450_cam) of P. putida (ATCC 17453) involved in camphor metabolism. The caffeine-inducible cytochrome P450-type enzyme exhibited a carbon monoxide difference spectrum peak at 450 nm, but does not allow growth on camphor. Caffeine induced production of haem-associated peroxidase activity was confirmed with 3,3, 5,5-tetramethylbenzidine–H₂O₂ reaction in polyacrylamide gels. Polymerase chain reaction (PCR) primers derived from the gene for cytochrome P450_cam (camC) of P. putida (ATCC 17453) did not yield an amplification product when DNA extracted from P. putida strain ATCC 700097 was used as template. The data demonstrate that caffeine is metabolized through a specific biphasic pathway driven by oxygen-demanding enzymes.     

The oxidatin of NADH by vanadate plus sugars

Sugars and sugar phosphates enable vanadate to catalyze the oxidation of NADH. Superoxide dismutase inhibits this oxidation. Incubation of sugars with vanadate, prior to addition of NADH, accelerates this oxidation of subsequently added NADH and eliminates the lag phase otherwise noted. Incubation of sugars with vanadate also results in the reduction of vanadate to vanadyl, with appearance of a blue-green color probably associated with a vanadyl-vanadate complex. It appears that sugars reduce vanadate to vanadyl which, in turn, reduces O₂ to O₂⁻ and that vanadate plus O₂⁻ then catalyzes the oxidation of NAD(P)H by a free radical chain reaction. Such oxidation of NAD(P)H may account for several of the biological effects of vanadate.     

Putative functions of phenylalanine-350 of Pseudomonas putida cytochrome P-450cam

Cytochrome P-450_cam hydroxylates d-camphor, using molecular oxygen and reducing equivalents transferred via putidaredoxin. We constructed mutant genes in which Phe-350 of P-450_cam was replaced by Leu, Tyr, or His by site-directed mutagenesis, expressed them in Escherichia coli, purified the mutant proteins, and compared their enzymic properties with those of the wild type P-450_cam. NADH oxidation rate of the Tyr mutant in the reconsituted system with putidaredoxin and putidaredoxin reductase was similar to that of the wild type enzyme, while the Leu mutant and the His mutant showed 67% and 17% activity of that of the wild type, respectively. The affinities of these mutant proteins for camphor and the oxidized form of putidaredoxin were much the same as those of the wild type protein. Rate constants for the reduction reaction of P-450_cam by reduced putidaredoxin, a physiological electron donor for P-450_cam, of Tyr and His mutants were much the same as that of the wild type enzyme, whereas the Leu mutant showed approx. half that of the wild type. Thus, the aromatic ring of Phe-350 of P-450_cam probably contributes to enhancing efficiency of the electron transfer yet does not seem to be essential for the reaction.     

Stepwise oxygenations of toluene and 4-nitrotoluene by a fungal peroxygenase

Fungal peroxygenases have recently been shown to catalyze remarkable oxidation reactions. The present study addresses the mechanism of benzylic oxygenations catalyzed by the extracellular peroxygenase of the agaric basidiomycete Agrocybe aegerita. The peroxygenase oxidized toluene and 4-nitrotoluene via the corresponding alcohols and aldehydes to give benzoic acids. The reactions proceeded stepwise with total conversions of 93% for toluene and 12% for 4-nitrotoluene. Using H₂¹⁸O₂ as the co-substrate, we show here that H₂O₂ is the source of the oxygen introduced at each reaction step. A. aegerita peroxygenase resembles cytochromes P450 and heme chloroperoxidase in catalyzing benzylic hydroxylations.     

Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase

We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H₂O₂ with concurrent evolution of O₂. This pseudocatalatic degradation of H₂O₂ to O₂ is solely dependent on ascorbate and is blocked by a spin trap, α-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H₂O with subsequent regeneration of ascorbate by the reduction of MDA with H₂O₂ evolving O₂ through the intermediate formation of O₂⁻. Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN⁻ in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN⁻. Further kinetic studies indicate that SCN⁻ itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN⁻ blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H₂O₂ to O₂. Binding studies by optical difference spectroscopy indicate that SCN⁻ binds LmAPX (Kd = 100 ± 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN⁻ acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.     

Cytochrome P450 cam: SS- dimer and -SH derivative reactivities.

N-ethyl-maleimide alkylation converts ferric P450_cam to a (succinimido-cys)₄ protein with native optical and EPR spectra but insensitive to substrate induced shift of iron to high spin with Soret absorption to higher energy and inactive in putidaredoxin ferric-ferrous reduction. On photo or chemical reduction the ferrous protein oxygenates and, with reduced putidaredoxin, converts substrate to product. Mild oxidation of P450_cam yields a disulfide dimer whose properties on alkylation of 3 sulfhydryls equal the succinimido₄ monomer; additional alkylation converts either monomer or dimer to a P420.     

Microsensor Measurements of Sulfate Reduction and Sulfide Oxidation in Compact Microbial Communities of Aerobic Biofilms

The microzonation of O₂ respiration, H₂S oxidation, and SO₄^2- reduction in aerobic trickling-filter biofilms was studied by measuring concentration profiles at high spatial resolution (25 to 100 μm) with microsensors for O₂, S^2-, and pH. Specific reaction rates were calculated from measured concentration profiles by using a simple one-dimensional diffusion reaction model. The importance of electron acceptor and electron donor availability for the microzonation of respiratory processes and their reaction rates was investigated. Oxygen respiration was found in the upper 0.2 to 0.4 mm of the biofilm, whereas sulfate reduction occurred in deeper, anoxic parts of the biofilm. Sulfate reduction accounted for up to 50% of the total mineralization of organic carbon in the biofilms. All H₂S produced from sulfate reduction was reoxidized by O₂ in a narrow reaction zone, and no H₂S escaped to the overlying water. Turnover times of H₂S and O₂ in the reaction zone were only a few seconds owing to rapid bacterial H₂S oxidation. Anaerobic H₂S oxidation with NO₃^- could be induced by addition of nitrate to the medium. Total sulfate reduction rates increased when the availability of SO₄^2- or organic substrate increased as a result of deepening of the sulfate reduction zone or an increase in the sulfate reduction intensity, respectively.     

Peroxygenase reactions catalyzed by cytochromes P450

Cytochromes P450 (P450s) catalyze monooxygenation of a wide range of less reactive organic molecules under mild conditions. By contrast with the general reductive oxygen activation pathway of P450s, an H₂O₂-shunt pathway does not require any supply of electrons and protons for the generation of a highly reactive intermediate (compound I). Because the low cost of H₂O₂ allows us to use it in industrial-scale synthesis, the H₂O₂-shunt pathway is an attractive process for monooxygenation reactions. This review focuses on the P450-catalyzed monooxygenation of organic molecules using H₂O₂ as the oxidant.     

Pyridine as novel substrate for regioselective oxygenation with aromatic peroxygenase from Agrocybe aegerita

Agrocybe aegerita peroxidase (AaP) is a versatile extracellular biocatalyst that can oxygenate aromatic compounds. Here, we report on the selective oxidation of pyridine (PY) yielding pyridine N-oxide as sole product. Using H₂¹⁸O₂ as co-substrate, the origin of oxygen was confirmed to be the peroxide. Therefore, AaP can be regarded as a true peroxygenase transferring one oxygen atom from peroxide to the substrate. To our best knowledge, there are only two types of enzymes oxidizing PY at the nitrogen: bacterial methane monooxygenase and a few P450 monooxygenases. AaP is the first extracellular enzyme and the first peroxidase that catalyzes this reaction, and it converted also substituted PYs into the corresponding N-oxides.     

Cytochrome P450cin (CYP176A1) D241N: Investigating the role of the conserved acid in the active site of cytochrome P450s

P450_cin (CYP176A) is a rare bacterial P450 in that contains an asparagine (Asn242) instead of the conserved threonine that almost all other P450s possess that directs oxygen activation by the heme prosthetic group. However, P450_cin does have the neighbouring, conserved acid (Asp241) that is thought to be involved indirectly in the protonation of the dioxygen and affect the lifetime of the ferric-peroxo species produced during oxygen activation. In this study, the P450_cin D241N mutant has been produced and found to be analogous to the P450_cam D251N mutant. P450_cin catalyses the hydroxylation of cineole to give only (1R)-6β-hydroxycineole and is well coupled (NADPH consumed: product produced). The P450_cin D241N mutant also hydroxylated cineole to produce only (1R)-6β-hydroxycineole, was moderately well coupled (31 ± 3%) but a significant reduction in the rate of the reaction (2% as compared to wild type) was observed. Catalytic oxidation of a variety of substrates by D241N P450_cin were used to examine if typical reactions ascribed to the ferric-peroxo species increased as this intermediate is known to be more persistent in the P450_cam D251N mutant. However, little change was observed in the product profiles of each of these substrates between wild type and mutant enzymes and no products consistent with chemistry of the ferric-peroxo species were observed to increase.     

Involvement of Molecular Oxygen in the Enzyme-Catalyzed NADH Oxidation and Ferric Leghemoglobin Reduction

Ferric leghemoglobin reductase (FLbR) from soybean (Glycine max [L.] Merr) nodules catalyzed oxidation of NADH, reduction of ferric leghemoglobin (Lb⁺³), and reduction of dichloroindophenol (diaphorase activity). None of these reactions was detectable when O₂ was removed from the reaction system, but all were restored upon readdition of O₂. In the absence of exogenous electron carriers and in the presence of O₂ and excess NADH, FLbR catalyzed NADH oxidation with the generation of H₂O₂ functioning as an NADH oxidase. The possible involvement of peroxide-like intermediates in the FLbR-catalyzed reactions was analyzed by measuring the effects of peroxidase and catalase on FLbR activities; both enzymes at low concentrations (about 2 μg/mL) stimulated the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction. The formation of H₂O₂ during the FLbR-catalyzed NADH oxidation was confirmed using a sensitive assay based on the fluorescence emitted by dichlorofluorescin upon reaction with H₂O₂. The stoichiometry ratios between the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction were not constant but changed with time and with concentrations of NADH and O₂ in the reaction solution, indicating that the reactions were not directly coupled and electrons from NADH oxidation were transferred to Lb⁺³ by reaction intermediates. A study of the affinity of FLbR for O₂ showed that the enzyme required at least micromolar levels of dissolved O₂ for optimal activities. A mechanism for the FLbR-catalyzed reactions is proposed by analogy with related oxidoreductase systems.     

Cytochrome P450 as an oxene transferase.

In the presence of iodosobenzene, liver microsomes catalyze the O-dealkylation of 7-ethoxycoumarin. The reaction proceeds in the absence of NADPH and O₂ and is dependent on cytochrome P450. The results indicate that cytochrome P450 acts as an oxene transferase probably involving [FeO]³⁺ as the transient intermediate of active oxygen.     

Production of reactive oxygen species in decoupled, Ca2+-depleted PSII and their use in assigning a function to chloride on both sides of PSII

Extraction of Ca²⁺ from the oxygen-evolving complex of photosystem II (PSII) in the absence of a chelator inhibits O₂ evolution without significant inhibition of the light-dependent reduction of the exogenous electron acceptor, 2,6-dichlorophenolindophenol (DCPIP) on the reducing side of PSII. The phenomenon is known as “the decoupling effect” (Semin et al. Photosynth Res 98:235–249, 2008). Extraction of Cl^? from Ca²⁺-depleted membranes (PSII[–Ca]) suppresses the reduction of DCPIP. In the current study we investigated the nature of the oxidized substrate and the nature of the product(s) of the substrate oxidation. After elimination of all other possible donors, water was identified as the substrate. Generation of reactive oxygen species HO, H₂O₂, and O ₂ ^·? , as possible products of water oxidation in PSII(–Ca) membranes was examined. During the investigation of O ₂ ^·? production in PSII(–Ca) samples, we found that (i) O ₂ ^·? is formed on the acceptor side of PSII due to the reduction of O₂; (ii) depletion of Cl^? does not inhibit water oxidation, but (iii) Cl^? depletion does decrease the efficiency of the reduction of exogenous electron acceptors. In the absence of Cl^? under aerobic conditions, electron transport is diverted from reducing exogenous acceptors to reducing O₂, thereby increasing the rate of O ₂ ^·? generation. From these observations we conclude that the product of water oxidation is H₂O₂ and that Cl^? anions are not involved in the oxidation of water to H₂O₂ in decoupled PSII(–Ca) membranes. These results also indicate that Cl^? anions are not directly involved in water oxidation by the Mn cluster in the native PSII membranes, but possibly provide access for H₂O molecules to the Mn₄CaO₅ cluster and/or facilitate the release of H⁺ ions into the lumenal space.     

Implications of paraquat and hydrogen peroxide-induced oxidative stress treatments on the GABA shunt pathway in Arabidopsis thaliana calmodulin mutants

Arabidopsis mutants with T-DNA insertion in seven calmodulin genes (CAM) were used to determine the specific role of CAM in the tolerance of plants to oxidative stress induced by paraquat and hydrogen peroxide (H₂O₂) treatments. Arabidopsis calmodulin mutants (cam) were screened for seedling growth, seed germination, induced oxidative damage, and levels of γ-aminobutyric acid (GABA) shunt metabolites. Only the cam5-4 and cam6-1 mutants exhibited an increased sensitivity to paraquat and H₂O₂ during seed germination and seedling growth. In response to treatments with 3 μM paraquat and 1 mM H₂O₂, only the cam5-4, cam6-1 mutants showed significant changes in malonaldehyde (MDA) levels in root and shoot tissues, with highly increased levels of MDA. In terms of the GABA shunt metabolites, GABA was significantly elevated in root and shoot tissues in response to the paraquat treatments in comparison to alanine and glutamate, while the levels of all shunt metabolites increased in root tissue but not in the shoot tissue following the H₂O₂ treatments. GABA, alanine and glutamate levels were significantly increased in root and shoot of the cam1, cam4, cam5-4, and cam6-1 mutants in response to paraquat (0.5, 1 and 3 μM), while they were increased only in the root tissue of the cam1, cam4, cam5-4, and cam6-1 mutants in response to H₂O₂ (200 and 500 μM, 1 mM). These data show that the cam5-4 and cam6-1 mutants were sensitive to the induced oxidative stress treatments in terms of seed germination, seedling growth, and oxidative damage. The accumulation of GABA shunt metabolites as a consequence of the induced oxidative stress treatments (paraquat and H₂O₂ treatments) suggests that the GABA shunt pathway and the accumulation of GABA metabolites may contribute in antioxidant machinery associated with reactive oxygen species and in the acquisition of tolerance in response to induced oxidative stress in Arabidopsis seedlings.     

The mechanism of photosynthetic water oxidation

Photosynthetie water oxidation is unique to plants and cyanobacteria, it occurs in thylakoid membranes. The components associated with this process include: a reaction center polypeptide, having a molecular weight (Mr) of 47–50 kilodaltons (kDa), containing a reaction center chlorophyll a labeled as P680, a plastoquinol(?)-electron donor Z, a primary electron acceptor pheophytin, and a quinone electron acceptor Q_A; three ‘extrinsic’ polypeptides having Mr of approximately 17 kDa, 23 kDa, and 33 kDa; and, in all likelihood, an approximately 34 kDa ‘intrinsic’ polypeptide associated with manganese (Mn) atoms. In addition, chloride and calcium ions appear to be essential components for water oxidation. Photons, absorbed by the so-called photosystem II, provide the necessary energy for the chemical oxidation-reduction at P680; the oxidized P680 (P680⁺), then, oxidizes Z, which then oxidizes the water-manganese system contained, perhaps, in a protein matrix. The oxidation of water, leading to O₂ evolution and H⁺ release, requires four such independent acts, i.e., there is a charge accumulating device (the so-called S-states). In this minireview, we have presented our current understanding of the reaction center P680, the chemical nature of Z, a possible working model for water oxidation, and the possible roles of manganese atoms, chloride ions, and the various polypeptides, mentioned above. A comparison with cytochrome c oxidase, which is involved in the opposite process of the reduction of O₂ to H₂O, is stressed. This minireview is a prelude to the several minireviews, scheduled to be published in the forthcoming issues of Photosynthesis Research, including those on photosystem II (by H.J. van Gorkom); polypeptides of the O₂-evolving system (by D.F. Ghanotakis and C.F. Yocum); and the role of chloride in O₂ evolution (by S. Izawa).     

The reaction of cysteine with ceruloplasmin copper

The kinetics of decay in absorbance at 610 nm in the reaction of cysteine with ceruloplasmin was biphasic under anaerobic conditions. Admission of oxygen to the bleached ceruloplasmin restored the blue color to about 75 % of the original value. However, under aerobic or anaerobic conditions an initial bleaching corresponded to a 25 % decrease in blue color. This change was irreversible and remained after removal of excess cysteine from the reaction mixture by dialysis. There was no correlation between transient and steady-state kinetic parameters. Circular dichroism measurements showed a characteristic reduction in the negative band at 450 nm, which is specific for type 1b copper. Isolation and further studies on cysteine-modified ceruloplasmin with a lower A₆₁₀/A₂₈₀ ratio showed < 10% reduction in enzyme activity toward p-phenylenediamine and o-dianisidine. Evidence is also presented that ceruloplasmin catalyzes the oxidation of cysteine with a one-electron reduction of oxygen and the formation of superoxide ion, which is then converted to H₂O₂ by ceruloplasmin. The effect of superoxide dismutase and catalase also confirms the presence of superoxide and H₂O₂. In sum, these data show that a permanent reduction of type 1b copper occurred when cysteine was used as a substrate. We conclude that there is a single electron transfer from cysteine directly to oxygen using one specific copper of ceruloplasmin, type 1b.