David C. Wharton

Identification of nitric oxide and nitrous oxide as products of nitrite reduction by Pseudomonas cytochrome oxidase (nitrite reductase)

Summary Both NO and N2O were identified by GC/MS as gaseous products of nitrite reduction catalyzed by the cytochrome oxidase (nitrite reductase) purified from Pseudomonas aeruginosa . Gas production was inhibited by cyanide. The enzyme also catalyzed the reduction of NO to N2O. No N2 was identified as a consequence of either nitrite or NO reduction.

Characterization of crystals of a cytochrome oxidase (nitrite reductase) from Pseudomonas aeruginosa by x-ray diffraction and electron microscopy.

Abstract Cytochrome oxidase from Pseudomonas aeruginosa has been crystallized from 2 m -ammonium sulfate. The crystals occur principally as thin diamond-shaped plates of space group P 2 1 2 1 2 with unit cell dimensions of 92 A × 115 A × 76 A. Determination of the density of glutaraldehyde-fixed, water-equilibrated crystals (1.167 g/cm 3 ), coupled with the unit cell volume (804,000 A 3 ), indicates that there is one subunit (~63,000 M r) per asymmetric unit. X-ray diffraction data which were limited to 12 A resolution due to small crystal size were obtained for the hk 0 and 0 kl zones using precession photography. Amplitude and phase data for the hk 0, 0 kl , and h 0 l zones were obtained from computer-based Fourier analysis of appropriate micrographs recorded from negatively stained microplates and thin sections of larger crystals using minimal beam electron microscopy. For crystals embedded in the presence of tannic acid it was possible to achieve 20 A resolution which is comparable to the resolution achieved with negative staining of thin crystalline arrays. In addition, unstained electron diffraction on glutaraldehyde-fixed, glucose-stabilized plates was recorded to a resolution of 9 A. The three-dimensional packing of the cytochrome oxidase dimer in the unit cell has been deduced from computer reconstructed images of the three principal projections along the crystallographic axes. The cytochrome oxidase dimer is located in the unit cell with the dimer axis coincident with a crystallographic 2-fold axis; thus within the resolution of the present data in projection (9 A) the two subunits are identical, in agreement with biochemical evidence. The crystals have been prepared with the enzyme in the fully oxidized state and upon reduction a progressive cracking of the crystals is observed, possibly due to a conformational change dependent on the oxidation state of the heme iron.

Cytochrome oxidase from Pseudomonas aeruginosa. IV. Reaction with oxygen and carbon monoxide.

The reaction between a cytochrome oxidase from Pseudomonas aeruginosa and oxygen has been studied by a rapid mixing technique. The data indicate that the heme d1 moiety of the ascorbate-reduced enzyme is oxidized faster than the heme c component. The oxidation of heme d1 is accurately second order with respect to oxygen and has a rate constant of 5.7 - 10(4) M-1 - s-1 at 20 degrees C. The oxidation of the heme c has a first order rate constant of about 8 s-1 at infinite concentration of O2. The results indicate that the rate-limiting step is the internal transfer of electrons from heme c to heme d1. These more rapid reactions are followed by more complicated but smaller abcorbance changes whose origin is still not clear. The reaction of ascorbate-reduced oxidase with CO has also been studied and is second order with a rate constant of 1.8 - 10(4) M-1 - s-1. The initial reaction with CO is followed by a slower reaction of significantly less magnitude. The equilibrium constant for the reaction with CO, calculated as a dissociation constant from titrimetric experiments with dithionite-reduced oxidase, is about 2.3 - 10(-6) M. From these data a rate constant of 0.041 s-1 can be calculated for the dissociation of CO from the enzyme.

Heme c - heme d1 interaction in Pseudomonas cytochrome oxidase (nitrite reductase): a reappraisal of the spectroscopic evidence.

Abstract Magnetic circular dichroism (MCD) spectra of Pseudomonas aeruginosa cytochrome oxidase are reported over the spectral range of 350–700 nm for the oxidized, ascorbate-reduced, dithionite-reduced and reduced carbon monoxide forms. The spectra of all forms examined can be interpreted as the simple sum of the individual heme c and heme d1 contributions without invoking “heme-heme interaction.” In particular and contrary to a recent report [Orii, Shimada, Nozawa, and Hatano, this Journal 76 , 983 (1977)] no effect of ligand binding to ferrous heme d1 was observed in the MCD spectrum of the heme c component. It seems likely that the previous findings were the result of incomplete reduction of the enzyme in the absence of stabilizing ligands.

Small-angle X-ray scattering studies of oxidized and reduced cytochrome oxidase from Pseudomonas aeruginosa

Small-angle X-rays scattering experiments were performed with oxidized and reduced cytochrome oxidase purified from Pseudomonas aeruginosa. The radii of gyration were calculated to be 40.5 A for the oxidized form and 37.0 A for the reduced. The longest dimension of the oxidized enzyme was 120 A while for the reduced it was 100 A. The volume of the oxidized protein was observed to be slightly greater than that of the reduced. These data indicate that there is a contraction of the structure of the enzyme during reduction of its constituent heme groups.

Detection of conformational changes in Complex III of the respiratory chain by a maleimido spin label

Changes in the conformation of Complex III (CoQH2-cytochromec reductase) of the mitochondrial respiratory chain were detected upon oxidoreduction using the nitroxide spin label, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl. EPR spectra of the spin label show a transition from a greater to a lesser degree of immobilization when the labeled enzyme, reduced either with ascorbate or sodium dithionite, is oxidized with potassium ferricyanide or ferricytochromec. These observations are interpreted to indicate that Complex III is more compact in the reduced state at least in the locality of the spin label. An apparent increase in the concentration of total spins during oxidation of the complex suggests change in the interaction between the spin label and other paramagnetic centers and not an oxidation of spin label, itself, since reduced free spin label could not be reoxidized. Addition of antimycin A had no effect on the EPR spectrum of the spin-labeled enzyme, indicating that this inhibitor does not initiate a conformational change in the region of the spin label. Experiments in which N-ethyl-[2-3H] maleimide was bound to Complex III show that binding occurs primarily to a subunit with a molecular weight of 45,000. Although no qualitative differences were observed, it was found that less radioactivity appears in samples reduced with dithionite than in those reduced with ascorbate. This difference appears to be caused by decomposition products of dithionite.

The use of a maleimido spin label to detect conformational changes in cytochrome c oxidase.

Abstract Spin labeling with a maleimido spin label has been used to investigate conformational changes of bovine cytochrome c oxidase. These experiments show that the spin label is immobilized to a lesser degree when the enzyme is in the “oxygenated” form than it is in the oxidized state and support the view that the oxygenated form is a conformational variant. Experiments in which the maleimido spin-labeled cytochrome c oxidase was titrated with H2O2 reveal that the peroxide-treated enzyme, although possessing an absorption spectrum similar to that of the oxygenated form, has an electron paramagnetic resonance (epr) spectrum that is different from that of either the oxygenated form or the oxidized state. Extremes of pH cause a marked decrease in the degree of immobilization of maleimido spin labels bound to the oxidase. Alterations in the epr spectrum are reversible if the pH is held between 5.3 and 10.2 but are irreversible outside that range. Urea and guanidine hydrochloride also decrease the immobilization of the spin labels bound to the oxidase. The nature of the epr spectra indicates that under these conditions the enzyme assumes a more open conformation. Exposure to concentrations of sodium dodecyl sulfate as high as 10% does not result in as much loss of the immobilization as with urea or guanidine. Detergents such as cholate, Tween 80, and Triton X-100 have no significant effect on the epr spectrum of maleimido spin-labeled cytochrome c oxidase.

Perturbation of Pseudomonas cytochrome oxidase by guanidine hydrochloride to detec differential stabilization of the heme d1 and heme c moieties

The optical properties of Pseudomonas cytochrome oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.2) were monitored as a function of guanidine hydrochloride (Gdn X HCl) concentration to probe for differential stabilization of its prosthetic groups, heme d1 and heme c. The protein fluorescence intensity increased with the Gdn X HCl concentration, revealing two transitions, a sharp one between 1.3 and 1.5 M Gdn X HCl, and a second less well defined extending from 2.5 to 4.5 M. Only the transition at the lower Gdn X HCl concentrations was present in titrations followed using the emission maxima. The spectral maximum for native Pseudomonas cytochrome oxidase was at approx. 335 nm and shifted to approx. 350 nm above 2 M Gdn X HCl. The heme d1 absorbance at 638 nm decreased with increasing [Gdn X HCl], giving a transition at 1.3-1.5 M, and no transition up to 4 M Gdn X HCl when the heme c was monitored at 525 nm. Along with the decrease at 638 nm, an absorption band appeared at 681 nm, suggesting heme d1 release into solution. Fluorescence titration of heme d1-depleted enzyme, prepared by gel filtration, showed a single transition similar to the transition occurring in the intact enzyme at high Gdn X HCl concentrations. Circular dichroism spectra revealed clearly distinguishable transitions for the heme d1 and heme c near 1.5 and 3.0 M Gdn X HCl, respectively. These results suggest that the two hemes are in regions of the protein with different stabilities which may represent distinct structural domains.