Pseudomonas Cytochrome Oxidase Research Articles

Preparation and spectral characterization of the heme d1 · apomyoglobin complex: An unusual protein environment for the substrate-binding heme of Pseudomonas cytochrome oxidase

The heme d1 prosthetic group isolated from Pseudomonas cytochrome oxidase combines with apomyoglobin to form a stable, optically well-defined complex. Addition of ferric heme d1 quenches apomyoglobin tryptophan fluorescence suggesting association in a 1:1 molar ratio. Optical absorption maxima for heme d1.apomyoglobin are at 629 and 429 nm before, and 632 and 458 nm after dithionite reduction; they are distinct from those of heme d1 in aqueous solution but more similar to those unobscured by heme c in Pseudomonas cytochrome oxidase. Cyanide, carbon monoxide and imidazole alter the spectrum of heme d1.apomyoglobin demonstrating axial coordination to heme d1 by exogeneous ligands. The cyanide-induced optical difference spectra exhibit isosbestic points, and a Scatchard-like analysis yields a linear plot with an apparent dissociation constant of 4.2 X 10(-5) M. However, carbon monoxide induces two absorption spectra with Soret maxima at 454 or 467 nm, and this duplicity, along with a shoulder that correlates with the latter before binding, suggests multiple carbon monoxide and possibly heme d1 orientations within the globin. The 50-fold reduction in cyanide affinity over myoglobin is more consistent with altered heme pocket interactions than the intrinsic electronic differences between the two hemes. However, stability of the heme d1.apomyoglobin complex is verified further by the inability to separate heme d1 from globin during dialysis and column chromatography in excess cyanide or imidazole. This stability, together with a comparison between spectra of ligand-free and -bound derivatives of heme d1-apomyoglobin and heme d1 in solution, implies that the prosthetic group is coordinated in the heme pocket through a protein-donated, strong-field ligand. Furthermore, the visible spectrum of heme d1.apomyoglobin varies minimally with ligand exchange, in contrast to the Soret, which suggests that much spectral information concerning heme d1 coordination in the oxidase is lost by interference from heme c absorption bands. A comparison of the absorption spectra of heme d1.apomyoglobin and Pseudomonas cytochrome oxidase, together with a critical examination of the previous axial ligand assignments from magnetic resonance techniques in the latter, implies that it is premature to accept the assignment of bishistidine heme d1 coordination in oxidized, ligand-free oxidase and other iron-isobacteriochlorin-containing enzymes.

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 in guanidine hydrochloride (Gdn · HCl) concentration to probe for differential stabilization of its prosthetic groups, heme d 1 and heme c. The protein fluorescence intensity increased with the Gdn · HCl concentration, revealing two transitions, a sharp one between 1.3 and 1.5 M Gdn · HCl, and a second less well defined extending from 2.5 to 4.5 M. Only the transition at the lower Gdn · 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 · HCl. The heme d 1 adsorbance at 638 nm decreased with increasing [Gdn · HCl], giving a transition at 1.3–1.5 M, and no transition up to 4 M Gdn · 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 d 1 release into solution. Fluorescence titration of heme d 1-depleted enzyme, prepared by gel filtration, showed a single transition similar to the transition occurring in the intact enzyme at high Gdn· HCl concentrations. Circular dichroism spectra revealed clearly distinguishable transitions for the heme d 1 and heme c near 1.5 and 3.0 M Gdn · HCl, respectively. There results suggest that the two hemes are in regions of the protein with different stabilities which may represent distinct structural domains.

Variations in the oxidation-reduction behavior of liganded species of Pseudomonas cytochrome oxidase.

In an effort to determine the steady-state redox properties of Pseudomonas aeruginosa cytochrome cd1, changes in absorption spectra after the addition of excess reductant (ascorbate, ferrous ethylenediaminetetraacetic acid) were monitored for degassed unliganded enzyme and samples in the presence of CO and CN- at pH 6.0, 8.0, or 10.0. Plots of [c2+]/[c3+] vs. [d2+]/[d3+] indicate that a "pseudoequilibrium" was reached for all samples at pH 8.0. Calculated values of delta Ed-c, the difference in reduction potential between the heme c and heme d moieties, at pH 8.0 were -25 +/- 5 (unliganded), -10 +/- 5 (enzyme-CO), and -25 +/- 5 mV (enzyme-CN). Relative rates of heme c and heme d reduction were found to be dependent upon type of ligand, reductant, and pH. Evidence for a cooperative heme c-heme d interaction is discussed.

Purification of Pseudomonas cytochrome oxidase (or nitrite reductase) by immunological methods

A new purification procedure for the cytochrome oxidase from Pseudomonas aeruginosa based on immunoaffinity chromatography has been compared with the biochemical method and shown to be (i) fully competitive in terms of chemical homogeneity and enzymatic properties of the purified protein (ii) slightly less efficient in terms of total recovery and (iii) much more convenient in terms of the time required. A further evolution of the method that minimizes the number of purification steps and any stress to the native structure of the protein is suggested.

Electron paramagnetic resonance study of the interaction of some anionic ligands with oxidized Pseudomonas cytochrome oxidase.

Electron paramagnetic resonance study of the interaction of some anionic ligands with oxidized Pseudomonas cytochrome oxidase.

Activation of Pseudomonas cytochrome oxidase by limited proteolysis with subtilisin.

Oxidized Pseudomonas cytochrome oxidase (ferrocytochrome c2: oxygen oxidoreductase; E.C.1.9.3.2) can be digested with subtilisin under controlled conditions that convert the original parent polypeptide chain (Mr on SDS gels approximately equal to 60,000) to a slightly smaller species (Mr on SDS gels approximately equal to 58,000). Under the conditions used (0.33% subtilisin, w/w, pH 7.4), the product formed from the oxidase was relatively stable to further digestion. Cytochrome oxidase activity was assayed at intervals during proteolysis by following the rate of oxidation of Pseudomonas ferrocytochrome c-551 by the enzyme in the presence of oxygen. The activity increased to a plateau that was more than two times the value for an untreated control. These observations suggest that clipping a small peptide from Pseudomonas cytochrome oxidase either facilitates the rate-limiting electron transfer between the intraprotein heme c and heme d1, enhances the interaction of the enzyme with ferrocytochrome c-551, or both.

Resolution of two distinct electron transfer sites on azurin.

Pseudomonas aeruginosa azurin is stoichiometrically and specifically labeled upon reduction by Cr(II)aq ions, yielding a substitution-inert Cr(III) adduct on the protein surface. We investigated the effect of this chemical modification on the reactivity of azurin with two of its presumed partners in the redox system of the bacterium. The Pseudomonas cytochrome oxidase catalyzed oxidation of reduced native and Cr(III)-labeled azurin by O2 was found to be unaffected by the modification. The kinetics of the electron exchange reaction between native or Cr(III)-labeled azurin and cytochrome c551 were studied by the temperature-jump method. Though similar chemical relaxation spectra were observed for native and modified systems, they differ quantitatively. Analysis of the concentration dependences of the relaxation times and amplitudes showed that both obey the same mechanism but that the specific reaction rates of the Cr(III)-modified protein are attenuated. This decreased reactivity of Cr(III)-labeled azurin toward one of its physiological partners suggests the involvement of the labeled region in the electron transfer reaction with cytochrome c551. Furthermore, the presence of a second active site, involved in the reduction of cytochrome oxidase, is suggested by the results.

Controlled proteolysis by subtilisin as a probe for cyanide-induced conformational changes in Pseudomonas cytochrome oxidase.

Oxidized Pseudomonas cytochrome oxidase (ferrocytochrome c-551:oxidoreductase; EC 1.9.3.2), digested with subtilisin in the absence and presence of KCN, produces discrete, high molecular weight fragments. The presence of KCN alters the rate of this fragmentation but does not change the nature of the fragments. When digested in the absence of KCN, the oxidase gives a major product (A) which is enzymatically active and has an apparent Mr = 58,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the presence of KCN, the major product (B) has Mr = 48,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis but with gel permeation high performance liquid chromatography it has an apparent Mr = 92,000. This implies that product B is dimeric, as is the parent enzyme which has Mr = 110,000 by high performance liquid chromatography. Absorption spectra of product B, isolated by gel filtration, show that it contains only the heme d1 moiety. The digestion time course indicates that the rate at which several minor products are formed is also dependent on the absence or presence of KCN. These observations suggest that the binding of KCN to the heme centers induces a conformational change in the enzyme so that the heme c-containing portion of the protein, which is at one end of the intact enzyme, can be removed without disrupting the integrity of the heme d1-containing portion.

Resonance Raman spectra of heme c and heme d1 in Pseudomonas cytochrome oxidase

Resonance Raman spectra of heme c and heme d1 in Pseudomonas cytochrome oxidase

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.

EPR study of heme x NO complexes of ascorbic acid-reduced Pseudomonas cytochrome oxidase and corresponding model complexes.

The EPR spectra of the NO complexes of frozen solutions of ascorbic acid-reduced cytochrome oxidase (nitrite reductase) purified from Pseudomonas aeruginosa, of its heme d1-depleted form, and of heme d1 in solutions containing various nitrogenous bases are quite similar to each other as well as to several heme (iron protoporphyrin IX)-containing proteins. The NO complexes of heme d1 (an iron-chlorin) in the presence of nitrogenous bases belong to spectral type C according to Kon's classification and, thus, the energy levels of the iron are closely related to thorse of heme complexes recorded under similar conditions. Comparison of these spectra with those of complexes of known structure suggests that both heme c and heme d1 are linked with Pseudomonas cytochrome oxidase by means of a nitrogenous ligand. The EPR spectrum of the NO complex of the native enzyme exhibits a lack of resolution of the high field (gy) resonance which can be characterized in terms of a spectral contribution from both the heme c and heme d1 moieties. The similarity between the EPR spectra of the NO complexes of horse heart cytochrome c and the heme d1-depleted Pseudomonas cytochrome oxidase before and after interaction with urea suggests structural similarities involving the heme irons. The changes caused by urea are likely to be a breaking or distortion of the bond between the iron and the protein-donated nitrogenous ligand and are similar to alterations seen with NO complexes of hemoglobin under a variety of conditions.

A re-evaluation of some basic structural and functional properties of Pseudomonas cytochrome oxidase.

Determinations of iron content and dry-weight measurements on samples of Pseudomonas cytochrome oxidase were coupled with sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis studies of both the native protein and covalently cross-linked oligomers in order to estimate the enzyme's molecular weight and spectral absorption coefficients. A value of epsilon(ox.) (410)=282x10(3) litre.mol(-1).cm(-1) was calculated for a dimeric protein molecule having a total molecular weight of 122000 (based on iron analysis). Steady-state kinetic observations of the enzyme-catalysed oxidation of reduced azurin by nitrite indicated a marked increase in enzyme inactivation as the pH was raised from 5.7 to 7.2. Since NO, a product of the nitrite reductase activity of Pseudomonas cytochrome oxidase, is known to bind to the enzyme, a study was undertaken to try to assess the potential of NO as a product inhibitor. Investigations showed that samples of the oxidized protein at pH values 4, 5 and 6 bound NO to both haem c and d(1) components, but oxidized enzyme samples at pH7 and above formed their reduced ligand-bound forms when placed under an atmosphere of the gas. Ascorbate-reduced enzyme samples at pH4, 5, 6 and 7 were also found to bind NO at both haem components, although at pH7 the rate of haem c binding was very slow. At pH8 and 9 only the ferrohaem d(1) bound NO. Titration experiments on the reduced protein over the pH range 5-7, with nitrite as a precursor of NO, showed that the haem d(1) had a much higher affinity than the haem c: experiments at pH5.2 and 5.9 with NO-equilibrated solutions revealed the same pattern of behaviour with the oxidized enzyme.

Allosteric cooperative interactions among redox sites of Pseudomonas cytochrome oxidase

Anaerobic reductive spectrophotometric titrations of Pseudomonas aeruginosa cytochrome oxidase were performed. Both types of hemes (C and D) of the dimeric enzyme were monitored. The reduction process was found to involve cooperative allosteric and spectroscopic interactions between the two subunits. The model fitting the data best involves the following features. (1) The redox potential of heme C is about 60 mV higher than that of heme D. (2) In the electron uptake, a positive cooperativity of about 30 mV exists between the two D-type hemes residing in the two subunits. (3) A negative cooperativity of the same magnitude (30 mV) is found between the two C-type hemes bound to two subunits. (4) No interaction was found between heme C and D in the same subunit or in the different subunits. (5) It is suggested that the reduction of the heme, of each kind, has about twice the spectral change compared to that observed upon reduction of the second one. The possible significance of this model for the mechanism of action of the enzyme is discussed

Some magnetic properties of Pseudomonas cytochrome oxidase

The magnetic properties of the haem groups of Pseudomonas cytochrome oxidase and its cyanide-bound derivatives were studied in both the oxidized and reduced states by means of m.c.d. (magnetic circular dichroism) at low temperatures. In addition, the oxidized forms of the enzyme were also investigated by e.p.r. (electron-paramagnetic-resonance) spectroscopy, and a parallel study, using both e.p.r. and m.c.d., was made on Pseudomonas cytochrome c-551 to aid spectral assignments. For ascorbate-reduced Pseudomonas cytochrome oxidase, the temperature-independence of those features in the m.c.d. spectrum corresponding to the haem c, and the temperature-dependence of those signals corresponding to the haem d1, showed the former to be low-spin and the latter to be high-spin (s = 2). However, addition of cyanide to the reduced enzyme gave a form of the protein that was completely low-spin. The e.p.r. and m.c.d. sectra of oxidized Pseudomonas cytochrome oxidase and its cyanide derivative were consistent with the haem c and d1 components being low-spin in both cases. Pseudomonas cytochrome c-551 was found to be low-spin in both its oxidized and reduced redox states.

The reactions of Pseudomonas cytochrome c-551 oxidase with potassium cyanide.

The binding of cyanide to both oxidized and ascorbate-reduced forms of Pseudomonas cytochrome c-551 oxidase was investigated. Spectral studies on the oxidized enzyme and its apoprotein showed that the ligand can bind to both the c and d, haem components of the molecule, and kinetic observations indicated that both chromophores reacted, under a variety of conditions, with very similar rates. Cyanide combination velocities were dependent on ligand concentration, and increasing the pH also accelerated the reaction; the second-order rate constant was estimated as approx. 0.2M-1 . s-1 at pH 7.0. The binding of cyanide to the protein was observed to have a considerable influence on reduction of the enzyme by ascorbate. Spectral and kinetic observations have revealed that the species haem d13+-cyanide and any unbound haem c may react relatively rapidly with the reductant, but the behaviour of cyanide-bound haem c indicates that it may not be reduced without prior dissociation of the ligand, which occurs relatively slowly. The reaction of reduced Pseudomonas cytochrome oxidase with cyanide is radically different from that of the oxidized protein. In this case the ligand only binds to the haem d1 component and reacts much more rapidly. Stopped-flow kinetic measurements showed the binding to be biphasic in form. Both the rates of these processes were dependent on cyanide concentration, with the fast phase having a second-order rate constant of 9.3 X 10(5) M-1 . s-1 and the slow phase one of 2.3 X 10(5) M-1 . s-1. The relative proportions of the two phases also showed a dependency on cyanide concentration, the slower phase increasing as the cyanide concentration decreased. Computer simulations indicate that a reaction scheme originally proposed for the reaction of the enzyme with CO is capable of providing a reasonable explanation of the experimental results. Static-titration data of the reduced enzyme with with cyanide indicated that the binding was non-stoicheiometric, the ligand-binding curve being sigmoidal in shape. A Hill plot of the results yielded a Hill coefficient of 2.6.

The oxidation of Pseudomonas cytochrome c-551 oxidase by potassium ferricyanide

Stopped-flow kinetics were made of the reaction between ascorbate-reduced Pseudomonas cytochrome oxidase and potassium ferricyanide under both N2 and CO atmospheres. Under N2 three kinetic processes were observed, two being dependent on ferricyanide concentration, with second-order rate constants of 9.6 X 10(4)M-1.s-1 and 1.5 X 10(4)M-1.s-1, whereas the other was concentration-independent, with a first-order rate constant of 0.17 +/- 0.03s-1. Measurements of their kinetic difference spectra have allowed the fastest and second-fastest phases of the reaction to be assigned to direct bimolecular reactions of ferricyanide with the haem c and haem d, moieties of the enzyme respectively. Under CO, the second-order rate constant for the reaction of the haem c was, at 1.3 X 10(5)M-1.s-1, slightly enhanced over the rate in a N2 atmosphere, but the reaction velocity of the haem d1 component was greatly decreased, being apparently limited to that of the rates of CO dissociation from the molecule (0.15s-1 and 0.03s-1). The results are compared with those obtained during a previous study of the reaction of reduced Pseudomonas cytochrome oxidase with oxidized azurin.

The reaction of Pseudomonas aeruginosa cytochrome c-551 oxidase with oxygen

The reaction of ascorbate-reduced Pseudomonas cytochrome oxidase with oxygen was studied by using stopped-flow techniques at pH 7.0 and 25 degrees C. The observed time courses were complex, the reaction consisting of three phases. Of these, only the fastest process, with a second-order rate constant of 3.3 X 10(4) M-1.S-1, was dependent on oxygen concentration. The two slower processes were first-order reactions with rates of 1.0 +/- 0.4s-1 and 0.1 +/- 0.03s-1. A kinetic titration experiment revealed that the enzyme had a relatively low affinity constant for oxygen, approx. 10(4)M-1. Kinetic difference spectra were determined for all three reaction phases, showing each to have different characteristics. The fast-phase difference spectrum showed that changes occurred at both the haem c and haem d1 components of the enzyme during this process. These changes were consistent with the haem c becoming oxidized, but with the haem d1 assuming a form that did not correspond to the normal oxidized state, a situation that was not restored even after the second kinetic phase, which reflected further changes in the haem d1 component. The results are discussed in terms of a kinetic scheme.

Association of Pseudomonas cytochrome oxidase with liposomes

Purified Pseudomonas cytochrome oxidase has been associated with asolectin liposomes by two different methods. Firstly, the enzyme was attached to liposomic membranes by adding it to a cholate-phospholipid dispersion and subsequently dialyzing the detergent out of suspension. In the second case the enzyme was adsorbed on the preformed liposomes when added to them after the dialysis. A stimulation of the cytochrome oxidase activity approximately twenty-fold was observed by the first method. In contrast, the activation was absent in the second type of preparation, indicating that interaction between the enzyme and phospholipids is very different in the two types of vesicles. The cholate-dialysis method for reconstitution of protein-phospholipid vesicles seems to lead to rather heterogeneous preparations. These can be further fractionated, not only according to their size but also to the protein/phospholipid ratio, by gel chromatography.

The quaternary structure of Pseudomonas cytochrome oxidase studied by electron microscopy

Pseudomonas cytochrome oxidase (EC 1.9.3.2) was studied by negative staining in the electron microscope. The best resolution was obtained with uranyl oxalate (pH 6.0) as negative stain. Electron micrographs confirm the idea of the dimeric structure of the enzyme. A rough model of cytochrome oxidase was constructed based on different projections of the molecule seen in the electron micrographs. In this model the subunits are identical and sterically equivalent.

A purification procedure for the soluble cytochrome oxidase and some other respiratory proteins from Pseudomonas aeruginosa

The production of the soluble cytochrome oxidase/nitrite reductase in the bacterium Pseudomonas aeruginosa is favoured by anaerobic conditions and the presence of KNO3(20g/l) in the culture medium. Of three methods commonly used for the disruption of bacterial suspensions (ultrasonication, liquid-shear homogenization and glass-bead grinding), sonication proved the most efficient in releasing the Pseudomonas cytochrome oxidase. A polarographic assay of Pseudomonas cytochrome oxidase activity with sodium ascorbate as substrate and NNN'N'-tetramethyl-p-phenylenediamine dihydrochloride as electron mediator is described. A purification procedure was developed which can be used on the small scale (40-litre cultures) or the large scale (400-litre cultures) and provides high yields of three respiratory-chain proteins, Pseudomonas cytochrome oxidase, cytochrome c551 and azurin, in a pure state. A typical preparation of 250g of Ps.aeruginosa cell paste yielded 180mg of Pseudomonas cytochrome oxidase, 81 mg of Pseudomonas cytochrome c551 and 275mg of Pseudomonas azurin.