The structure of the paramagnetic oxygen intermediate in the cytochrome c oxidase reaction.

A paramagnetic intermediate with an unusual e.p.r. spectrum is formed when fully reduced cytochrome c oxidase is allowed to react with dioxygen at 173 K. The effect on the e.p.r. spectrum of using dioxygen enriched in 17O was investigated. These experiments show that an oxygen atom derived from dioxygen is bound to Cu2+ in the intermediate. The e.p.r. parameters can be explained in terms of a weak antiferromagnetic interaction (J approximately equal to 10 cm-1) between Cu2+B and cytochrome a3 in the low-spin ferryl ion state. It is suggested that an OH- ion bound to Cu2+B is hydrogen bonded to the oxygen atom of the ferryl ion in cytochrome a3.     

Analysis of the electron paramagnetic resonance spectrum of the cobalamin intermediate in ribonucleotide reduction.

EPR absorption-derivative lineshapes have been computed and least-squares fitted to the spectrum of the intermediate derived from 5'-deoxy-5'-adenosylcobalamin in the ribonucleotide reductase reaction. A Gaussian-type intrinsic lineshape was assumed and the effects of inhomogenous broadening, rotation of coordinate axes of the A-tensor relative to the g-tensor, angular dependence of transition probability and ligand hyperfine splitting have also been investigated. When the overall spectrum was computed as the sum of the lineshapes corresponding to two distinct Co(II) species, A and B, each having rhombic symmetry, the least squares procedure converged to a much better fit than with a single species, and matched almost all of the features of the experimental spectrum. The magnetic properties of A and B were compared with those of a series of other Co(II) complexes by a plot of g - g versus A - A. The results eliminate cobalt with 5-coordination to nitrogen for A and B, and suggest low-spin cobalt complexes having strongly distorted 6-fold coordination. The possibility that the sixth, symmetry-decreasing ligand is the oxygen molecule is excluded by the chemistry of the system and by the EPR properties of previously reported cob(II)alamins. It is suggested that the sixth ligand is a carbonyl, amide or sulfhydryl group of an enzyme sidechain which is inserted off-axis into the coordination position so as to exert the observed symmetry-lowering effect.     

Analysis of the electron paramagnetic resonance spectrum of the cobalamin intermediate in ribonucleotide reduction

EPR absorption-derivative lineshapes have been computed and least-squares fitted to the spectrum of the intermediate derived from 5'-deoxy-5'-adenosyl-cobalamin in the ribonucleotide reductase reaction. A Gausian-type intrinsic lineshape was assumed and the effects of inhomogenous broadening, rotation of coordinate axes of the A-tensor relative to the g-tensor, angular dependence of transition probability and ligand hyperfine splitting have also been investigated.When the overall spectrum was computed as the sum of the linshapes corresponding to two distinct Co(II) species, A and B, each having rhombic asymmetry, the least squares procedure converged to a much better fit than with a single species, and matched almost all of the features of the experimental spectrum.The magnetic properties of A and B were compared with those of a series of other Co(II) complexes by a plot of g_|?g₆ versus ∥A₆∥?∥A_|∥. The results eliminate cobalt with 5-coordination to nitrogen for A and B, suggest low-spin cobalt complexes having strongly distorted 6-fold coordination. The possibility that the sixth, symmetry-decreasing ligand is the oxygen molecule is excluded by the chemistry of the system and by the EPR properties of previously reported cob(II)alamins. It is suggested that the sixth ligand is carbonyl, amide or sulfhydryl group of an enzyme sidechain which is inserted off-axis into the coordination position so as to exert the observed symmetry-lowering effect.     

Studies of the primary oxygen intermediate in the reaction of fully reduced cytochrome oxidase.

The formation and disappearance of a photosensitive species during the reaction of reduced cytochrome c oxidase (putatively a3II.O2), EC 1.9.3.1, has been followed by (a) mixing a3II.CO with O2 in a stopped flow apparatus; (b) initiating the oxygen-oxidase reaction by removing CO with a laser flash; (c) probing the reaction mixture for photosensitivity with a second laser flash. Photosensitivity appears in the reaction mixture after the first laser flash, reaches a maximum after 50-60 microseconds ([O2] greater than 100 microM), and disappears in a further 50-100 microseconds. The kinetics can be represented by the scheme [formula: see text]. In species B, O2 is associated with the protein, possibly CuB, but not with the heme. Species C is the photosensitive a3II.O2 complex, and in D, a3 iron has been oxidized. The formation of species C is responsible for the rapid phase of absorbance change in the oxidase-oxygen reaction. The rate of reaction with oxygen approaches the limit of 35,000 s-1 at high oxygen. Nitric oxide, however, reacts with FeII oxidase with a rate of 1 x 10(8) M-1 s-1, which is accurately maintained up to an observed rate of 10(5) s-1. In flash photolysis experiments, approximately half of the photodissociated nitric oxidase recombines in a biphasic geminate reaction with rates of 1 x 10(8) s-1 and 1 x 10(7) s-1.     

Kinetic studies of Rhus vernicifera laccase. Evidence for multi-electron transfer and an oxygen intermediate in the reoxidation reaction.

1. The reoxidation of reduced Rhus vernicifera laccase (monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) by molecular oxygen has been studied by optical absorption and EPR methods. 2. The reoxidation by oxygen of the type 1 Cu+ and the two-electron acceptor is characterized by a second-order rate constant of about 5-10(6) M-1-s-1. 3. The appearance of an optical intermediate (with an absorbance maximum around 360 nm) parallels the reoxidation of type 1 Cu+ and the two-electron acceptor. It disappears in a first-order reaction with a half-time of 20 s. A similar intermediate is formed during normal turnover. 4. The type 2 Cu+ appears to be reoxidized in an intramolecular reaction with a half-time of about 20 s, suggesting a correlation between the reoxidation of this site and the disappearance of the optical intermediate. 5. The results suggest that three electrons are rapidly transferred to oxygen leading to the formation of an enzyme-bound oxygen intermediate.     

Electron paramagnetic resonance studies of type 1 copper in type 2 depleted fungal laccase A

The electron paramagnetic resonance (EPR) spectra of type 1 copper(II) in 63Cu-enriched Coriolus versicolor laccase A (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) have been studied. The X-band EPR spectrum in type 2 copper-depleted [63Cu]laccase A exhibited well-resolved ligand superhyperfine structure in the g perpendicular region. This structure was assigned to an interaction with two nitrogens and two protons, an assignment which is consistent with a model in which the two nitrogens belong to two histidine ligands and the two protons are the methylene protons of a coordinating cysteine. It also requires the delocalization of a substantial amount of the type 1 copper(II) unpaired electron density onto the cysteine sulphur.     

Changes in intermediate haemoglobins during methaemoglobin reduction by NADPH-flavin reductase.

The changes in intermediate haemoglobins produced during methaemoglobin reduction by NADPH-flavin reductase were analysed by an isoelectric-focusing method. The alpha 3+ beta 2+ and alpha 2+ beta 3+ valency hybrids were observed as intermediate haemoglobins and changed consecutively with time during the reaction. On the basis of the analyses, the course of methaemoglobin reduction was found to involve two different pathways: (1) methaemoglobin kappa+1 leads to alpha 3+ beta 2+ kappa+2 leads to oxyhaemoglobin; (2) methaemoglobin kappa+3 leads to alpha 2+ beta 3+ kappa+4 leads to oxyhaemoglobin. The reaction rate constants of each phase (kappa+1--kappa+4) were also estimated. The addition of inositol hexaphosphate to the reaction mixture did not affect the overall reaction. The mechanism of methaemoglobin reduction by NADPH-flavin reductase is discussed on the basis of these results.     

EPR studies on the anaerobic reduction of fungal laccase. Evidence for participation of type 2 copper in the reduction mechanism.

1. In anaerobic reduction studies on fungal laccase B (p-diphenol:O2 oxidoreductase, EC 1.14.18.1) with the EPR and stopped-flow techniques it was found that the type 2 copper of the enzyme is rapidly undergoing a reduction-oxidation cycle which is followed by a slower reduction in a couple of seconds. An intermediate EPR signal of unknown origin is formed in the same time-range as the initial reduction of type 2 copper and disappears again when this copper ion is reoxidized. 2. The rate of the anaerobic reoxidation of type 2 copper is similar to the reduction rate of the two-electron acceptor, suggesting that they are interacting in the electron transfer of the enzyme. 3. The changes in the reaction rates of both type 2 and type 3 copper appear to be affected in a similar way by changes in pH. 4. The EPR signal of the type 2 Cu2+ suggests that this ion is liganded to one or more nitrogens.     

A new two-disulphide intermediate in the refolding of reduced bovine pancreatic trypsin inhibitor

The apparently complete refolding of reduced bovine pancreatic trypsin inhibitor (BPTI) is shown to produce a mixture of two species. One of these is native BPTI, but the other lacks the disulphide bond between cysteines 30 and 51. The latter species has a folded conformation very like that of native BPTI, and is oxidized by air to native BPTI on warming in aqueous solution. The two unreactive cysteine thiol groups appear to be buried in the interior of the molecule, which restricts access by reagents that can alkylate them or oxidize them to form the disulphide bond. The implications of this intermediate and its conformation for the understanding of protein folding are discussed.     

Periplasmic oxygen reduction by Desulfovibrio species

Washed cells of Desulfovibrio vulgaris strain Marburg (DSM 2119) reduced oxygen to water with H(2) as electron donor at a mean rate of 253 nmol O(2) min(-1) (mg protein)(-1). After separating the periplasm from the cells, more than 60% of the cytochrome c activity and 90% of the oxygen-reducing activity were found in the periplasmic fraction. Oxygen reduction and the reduction of cytochrome c with H(2) were inhibited by CuCl(2). After further separation of the periplasm by ultrafiltration (exclusion sizes 30, 50, and 100 kDa), oxygen reduction with H(2) occurred with the retentates only. Ascorbate plus tetramethyl-p-phenylenediamine (TMPD), however, were also oxidized by the filtrates. The stoichiometry of 1 mol O(2) reduced per 2 mol ascorbate oxidized indicated the formation of water. Our experiments present evidence that in D. vulgaris periplasmic hydrogenase and cytochrome c play a major role in oxygen reduction. Preliminary studies with other Desulfovibrio species indicated a similar function of periplasmic c-type cytochromes in D. desulfuricans CSN and D. termitidis KH1.     

Binding of nitric oxide to reduced L-tryptophan-2,3-dioxygenase as studied by electron paramagnetic resonance.

Ferrous L-tryptophan-2,3-dioxygenase reacts with nitric oxide both in the presence and in the absence of L-tryptophan. Electron paramagnetic resonance studies suggest that the proximal ligand of the heme is a nitrogen atom, probably from an histidyl residue. The interaction of the protein with substrate changes both the symmetry of the paramagnetic center and the mode of interaction of the iron atom with its two axial ligands, NO and the proximal nitrogen atom. Optical absorption and EPR spectra suggest that the affinity of NO for tryptophan dioxygenase increases in the order: tryptophan dioxygenase, tryptophan dioxygenase + alpha-methyltryptophan, tryptophan diogenase " 5-hydroxytryptophan, tryptophan dioxygenase + L-tryptophan. A possible correlation between the number of superhyperfine lines in the EPR spectrum and the affinity of the enzyme for NO is discussed.