Evidence for two H2O2-binding sites in ferric cytochrome c oxidase. Indication to the O-cycle?

H2O2 addition to the oxidized cytochrome c oxidase reconstituted in liposomes brings about a red shift of the Soret band of the enzyme and an increased absorption in the visible region with two distinct peaks at approximately 570 and 605 nm. Throughout pH range 6-8.5, the spectral changes at 570 nm and in the Soret band titrate with very similar pH-independent Kd values of 2-3 microM. At the same time, Kd of the peroxide complex measured at 605 nm increases markedly with increased H+ activity reaching the value of 18 +/- 2 microM at pH 6.0. This finding may indicate the presence of two different H2O2-binding sites in the enzyme with different affinity for the ligand at acid pH. The Soret and 570 nm band effects are suggested to report H2O2 coordination to heme iron of alpha 3, whereas the maximum at 605 nm could arise from H2O2 binding to Cu alpha 3 followed by the enzyme transition into the 'pulsed' (or '420/605') conformation. Possible implication of the two H2O2-binding sites for the cytochrome oxidase redox and proton-pumping mechanisms are discussed.     

Formation and reduction of a ''peroxy'' intermediate of cytochrome c oxidase by hydrogen peroxide.

In the presence of micromolar concentrations of H2O2, ferric cytochrome c oxidase forms a stable complex characterized by an increased absorption intensity at 606-607 nm with a weaker absorption band in the 560-580 nm region. Higher (millimolar) concentrations of H2O2 result in an enzyme exhibiting a Soret band at 427 nm and an alpha-band of increased intensity in the 589-610 nm region. Addition of H2O2 to ferric cytochrome c oxidase in the presence of cyanide results in absorbance increases at 444nm and 605nm. These changes are not seen if H2O2 is added to the cyanide complex of the ferric enzyme. The results support the idea that direct reaction of H2O2 with ferric cytochrome a 3 produces a 'peroxy' intermediate that is susceptible to further reduction by H2O2 at higher peroxide concentrations. Electron flow through cytochrome a is not involved, and the final product of the reaction is the so-called 'pulsed' or 'oxygenated' ferric form of the enzyme.     

Proton interactions in the resting form of cytochrome oxidase.

The effect of pH on the near-UV absorption spectrum of cytochrome oxidase has been examined. Several lines of evidence implicate a proton binding site that can modulate the optical properties of cytochrome alpha 3 in the resting enzyme. Changing the pH within the range 6.5-10.5 was found to reversibly shift the position of the Soret band over an 11-nm range. The lower pH values caused a progressive blue shift in the Soret band, whereas the high-pH range promoted a gradual red shift. Limiting band positions were approximately 416 and 427 nm. The incubation time required to reach a stable band position varied somewhat as did the actual extent of the shift. In most cases, the shift was associated with an isosbestic point. A pH titration profile for the apparent equilibrium position of the Soret band was obtained. Nonlinear least-squares fitting to a scatter plot, assuming a single acid/base group, showed an apparent pKa of 7.8. Magnetic circular dichroism (MCD) spectra of the low-pH form at 416 nm, the high-pH form at 427 nm, and the cyanide derivative at 428 nm were compared. No evidence of a high-pH-dependent low-spin transition or a change in the redox state of cytochrome a3 was found, confirming earlier work [Baker, G. M., Noguchi, M., & Palmer, G. (1987) J. Biol. Chem. 262, 595-604]. Subtraction of ferricytochrome a [spectrum taken from Vanneste, W. H. (1966) Biochemistry 5, 838-848] from a series of blue-shifting spectra showed a band at 414 nm that progressively gained amplitude and a band at 430 nm that correspondingly lost amplitude. A series of red-shifting spectra showed the opposite behavior with a clear isosbestic point being evident in both cases. The difference extinction change at 414 and 430 nm depended linearly on the position of the Soret band, both showing a reversible dependence on pH. The 430-nm band is noted to be unusually red-shifted for high-spin ferric heme a. An additional, pH-insensitive band was observed at 408-410 nm which was eliminated by treatment with cyanide. The kinetics of the pH-induced blue shift and red shift were obtained at 416 nm by using dual-wavelength method and found to be biphasic, despite the occurrence of an isosbestic point.(ABSTRACT TRUNCATED AT 400 WORDS)     

The pH dependence of cytochrome a conformation in cytochrome c oxidase.

The pH dependence of the conformation of cytochrome a in bovine cytochrome c oxidase has been studied by second derivative absorption spectroscopy. At neutral pH, the second derivative spectra of the cyanide-inhibited fully reduced and mixed valence enzyme display two Soret electronic transitions, at 443 and 451 nm, associated with cytochrome a. As the pH is lowered these two bands collapse into a single transition at approximately 444 nm. pH titration of the cyanide-inhibited mixed valence enzyme suggests that the transition from the two-band to one-band spectrum obeys the Henderson Hasselbalch relationship for a single protonation event with a transition pKa of 6.6 +/- 0.1. No pH dependence is observed for the spectra of the fully reduced unliganded or CO-inhibited enzyme. Tryptophan fluorescence spectra of the enzyme indicate that no major disruption of protein structure occurs in the pH range 5.5-8.5 used in this study. Resonance Raman spectroscopy indicates that the cytochrome a3 chromophore remains in its ferric, cyanide-bound form in the mixed valence enzyme throughout the pH range used here. These data indicate that the transition observed by second derivative spectroscopy is not due simply to pH-induced protein denaturation or disruption of the cytochrome a3 iron-CN bond. The pH dependence observed here is in good agreement with those observed earlier for the midpoint reduction potential of cytochrome a and for the conformational transition associated with energy transduction in the proton pumping model of Malmstr?m (Malmstr?m, B. G. (1990) Arch. Biochem. Biophys. 280, 233-241). These results are discussed in terms of a model for allosteric communication between cytochrome a and the binuclear ligand binding center of the enzyme that is mediated by ionization of a single group within the protein.     

Kinetic studies on cytochrome c oxidase by combined epr and reflectance spectroscopy after rapid freezing.

1. Techniques and experiments are described concerned with the millisecond kinetics of EPT-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O2 or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy. 2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (less than 50 ms) approx. 0.5 electron equivalent per heme a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (less than 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a. 3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O2 in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O2 side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates. 4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms., whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO. 5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C.R., Hansen, R.E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477-2481). Both the low-spin (g=o; 2.2; 1.5) and slowly appearing high-spin (g=6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a3, when it is present in a state of interaction with EPR-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.     

Thermal denaturation of cytochrome c peroxidase: pH dependence

Upon heating cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) at pH 4 and 5, the enzyme precipitates at 41 degrees C and 51 degrees C, respectively. Incubating the enzyme at lower temperatures causes a slow dissociation of the heme from the protein. The heme precipitates, while the apoprotein remains soluble. Between pH 6 and 8, the native enzyme is converted to a low-spin ferric form upon heating. The Soret maximum shifts from 408 to 414 nm. The midpoint of this transition is pH-dependent, with a value of 46 degrees C at pH 6 decreasing to 29 degrees C at pH 8. At high temperatures the 414 nm form is converted to a species which has a 'free heme' spectrum with low absorptivity and Soret maximum at 390 nm. The midpoint temperature of this latter transition is 62 degrees C and 57 degrees C at pH 7 and 8, respectively.     

Yeast cytochrome c peroxidase. Coordination and spin states of heme prosthetic group

Electronic absorption and electron paramagnetic resonance (EPR) spectroscopic examinations revealed that a freshly prepared cytochrome c peroxidase (CCP) contains a penta-coordinated high spin ferric protoheme group. The penta-coordinated high spin state of fresh CCP is maintained in a remarkably wide range of pH (4-8). The freezing of fresh CCP induces the reversible coordination of an internal strong field ligand to the heme iron to form a hexa-coordinated low spin compound, which shows EPR extrema at gx = 2.70, gy = 2.20 and gz = 1.78. In the presence of glycerol the freezing-induced artifacts are eliminated and the fresh enzyme exhibits an EPR spectrum of rhombically distorted axial symmetry with EPR extrema at gx = 6.4, gy = 5.3, and gz = 1.97 at 10 K, characteristic of the penta-coordinated high spin enzyme. Upon aging CCP is converted to a hexa-coordinated high spin state due to the coordination of an internal weak field ligand to the heme iron. This conversion is accelerated at acidic pH values, and its reversibility varies from fully reversible to irreversible depending on the degree of enzyme aging. The aging-induced hexa-coordinated CCP is unreactive with hydrogen peroxide and exhibits an EPR spectrum of purely axial symmetry with extrema at g = 6 and g = 2 and an electronic absorption spectrum with an intensified Soret band at 408 nm (epsilon 408 nm = 120 mM-1 cm-1) and a blue-shifted charge-transfer band at 620 nm. Spectroscopic properties of different coordination and spin states of fresh and aged CCPs are compiled in order to formulate a generalized spectroscopic characterization of penta- and hexa-coordinated high spin ferric hemoproteins.     

Characterisation of 'fast' and 'slow' forms of bovine heart cytochrome-c oxidase.

We have prepared cytochrome-c oxidase from bovine heart (using a modification of the method of Kuboyama et al. (1972) J. Biol. Chem. 247, 6375-6383) which binds cyanide rapidly, shows no kinetic distinction between the two haems on reduction by dithionite, has a Soret absorption maximum above 424 nm, and has a negligible 'g' = 12' EPR signal. On incubation at pH 6.5 this 'fast' oxidase reverts to the 'slow' ('resting') form characterised by slow cyanide binding, slow reduction of haem a3 by dithionite, a blue-shifted Soret maximum and a large 'g' = 12' signal. Incubation of 'fast' oxidase with formate produces a form of the enzyme with properties almost identical to those of 'slow' oxidase. The kinetics of formate binding to 'fast' oxidase are found to be biphasic, revealing the presence of at least two 'fast' subpopulations in our preparations. Evidence is presented that there is an equilibrium mixture of high-spin and low-spin forms of haem a3 in both 'fast' subpopulations at room temperature. Incubation of 'fast' oxidase with chloride or bromide at pH 6.5 produces forms of oxidase with much lower rates of cyanide binding. Our working hypothesis is that formate mimics a binuclear centre ligand which is present in the 'slow' form of cytochrome oxidase. Although we show that chloride and bromide can also be ligands of the binuclear centre, possibly onto CuB, we can rule out either of these being the ligand present in the 'slow' enzyme. We will argue that the 'fast' and 'slow' forms of oxidase are equivalent to the 'pulsed' and 'resting' forms of oxidase, respectively.     

Effect of heat treatment on oxidase activity and proton-pumping capability of proteoliposome-incorporated beef heart cytochrome aa3

By incubating beef heart cytochrome c oxidase at 43-45 degrees C, selective inactivation of the H+-pumping function is possible without affecting cytochrome c oxidase activity; proteoliposomes reconstituted with heated enzyme (43.5 degrees C for 60 min at pH 7.0) showed an apparent H+/e- ratio of only 0.3 and a turnover with cytochrome c plus ferrocyanide as substrate of 20 s-1, while those with the intact enzyme showed an apparent H+/e- ratio somewhat greater than 1.0 and a turnover of 19 s-1. This decrease in the H+/e- ratio could not be attributed to a stimulation of H+ permeability upon heating, since the respiratory control ratio and the magnitude of membrane potential formation remained almost the same in the two cases. A pH-dependent Em (midpoint redox potential) change of cytochrome a in the presence of cyanide was still observed after the heat treatment. Heating induced a small spectral shift in the Soret region of the oxidized (resting) enzyme; the peak of the heated enzyme was at 421 nm, while that of the intact enzyme was at 419 nm. The spectral shift obtained by pulsing the enzyme with oxygen under turnover conditions is also altered.     

Oxidation of cytochrome c by cytochrome c oxidase: spectroscopic binding studies and steady-state kinetics support a conformational transition mechanism

The long-known biphasic response of cytochrome c oxidase to the concentration of cytochrome c has been explained, alternatively, by the presence of a catalytic and a regulatory site on the oxidase, by negative cooperativity between adjacent active sites in dimeric oxidase, or by a transition of the enzyme molecule between different conformational states. The three mechanistic hypotheses allow testable predictions about the relationship between substrate binding and steady-state kinetics catalyzed by the monomeric and dimeric (or oligomeric) enzyme. We have tested these predictions on monomeric, dimeric, and oligomeric beef heart oxidase and on monomeric oxidase from Paracoccus denitrificans. The aggregation state of the oxidase was evaluated from the sedimentation equilibrium in the ultracentrifuge and by gel chromatography. The binding of cytochrome c to cytochrome c oxidase was measured by spectrophotometric titration of cytochrome c oxidase with cytochrome c. The procedure makes use of a small perturbation in the Soret band of the absorption spectrum of the cytochrome c-cytochrome c oxidase complex. The steady-state oxidation of cytochrome c was followed spectroscopically by an automated assay procedure, and the kinetic parameters were deduced by numerical analysis of several hundred initial rate assays in the substrate concentration range 0.15-30 microM. The following results were obtained: (1) The kinetics of cytochrome c oxidation are always biphasic at low ionic strength, independent of the aggregation state of the enzyme. (2) The kinetics become apparently monophasic at ionic strengths above 100 mM or at slightly acidic pH values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Does the peroxide compound of cytochrome oxidase contain a ferryl iron?

The reaction of peroxide with cytochrome oxidase generates a peroxide compound having a Soret maximum at 428 nm. X-ray absorption spectroscopy analysis of the local structure of the active site iron shows marked similarity to that of the cytochrome c peroxidase intermediate Compound ES, which contains a short iron to proximal nitrogen distance compared to globins. Reductive titration of the 580 nm band of this compound indicates that the iron is one oxidizing equivalent above the resting oxidized form. These results support the presence of a ferryl iron (Fe(IV) = O) in the peroxide compound similar to that found for the peroxidases.     

Kinetic studies on cytochrome c oxidase by combined EPR and reflectance spectroscopy after rapid freezing

1. Techniques and experiments are described concerned with the millisecond kinetics of EPR-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O₂ or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy.2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (< 50 ms) approx. 0.5 electron equivalent per hame a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (> 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a.3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O₂ in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O₂ side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates.4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms, whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO.5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C. R., Hansen, R. E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477–2481). Both the low-spin (g = 3; 2.2; 1.5) and slowly appearing high-spin (g = 6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a₃, when it is present in a state of interaction with EPR-undetectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.     

Transient kinetic studies of Neurospora crassa cytochrome c oxidase.

The reaction of Neurospora crassa cytochrome c oxidase with CO was studied by flash-photolysis and rapid-mixing experiments, leading to the determination of the association and dissociation rate constants (7 X 10(4) M-1 X s-1 and 0.02s-1 respectively). Pre-steady-state kinetic investigations of the catalytic properties of the enzyme showed that under proper conditions Neurospora cytochrome c oxidase can be 'pulsed', i.e. activated, like the mammalian enzyme. The 'pulsed' species is spectroscopically different from the 'resting' one, and the decay into the 'resting' state is fast (t1/2 approx. 3 min).     

Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction of hydrogen peroxide with the rapid form of resting cytochrome oxidase.

The hydrogen peroxide binding reaction has been examined with alkaline-purified resting enzyme in order to avoid mixtures of low pH induced fast and slow conformers. At pH 8.8-9.0 (20 degrees C), the reactivity of resting enzyme was similar to the peroxide-free, pulsed conformer that has been characterized by other investigators. The reaction showed single-phase reactivity at 435 and 655 nm and required a minimum 8:1 molar excess of peroxide (over cytochrome a3) for quantitative reaction. At 16:1, the Soret band was stable for 1.0-1.5 h, but above 80:1, the band began showing generalized attenuation within 1-2 min. The peroxide binding reaction was also associated with an increase in absorbance at 606 nm which correlated with the rate of change at 435 and 655 nm. The observed rate constants at each of these wavelengths showed similar linear dependence on peroxide concentration, giving an average bimolecular rate constant of 391 M-1.s-1 and a Kd of 5.1 microM. The rise phase at 606 nm was observed to saturate at an 8:1 molar excess of peroxide but showed a slow, concentration-dependent first-order decay that gave a bimolecular rate constant and Kd of 38 M-1.s-1 and 20 microM, respectively. The decay was not associated with a change in the Soret absorption or charge-transfer regions, suggesting a type of spectral decoupling. An isosbestic point at 588 nm was consistent with the 606- to 580-nm conversion proposed by other investigators, although direct observation of a new band at 580 nm was difficult.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a catalase-peroxidase from the fungus Septoria tritici.

Three classes of heme proteins, commonly designated hydroperoxidases, are involved in the metabolism of hydrogen peroxide: catalases, peroxidases, and catalase-peroxidases. While catalases and peroxidases are widely spread in animals, plants, and microorganisms, catalase-peroxidases were characterized only in prokaryotes. We report here, for the first time, on a catalase-peroxidase in a eukaryotic organism. The enzyme was purified from the fungal wheat pathogen Septoria tritici, and is one of three different hydroperoxidases synthesized by this organism. The S. tritici catalase-peroxidase, designated StCP, is similar to the enzymes previously isolated from the bacteria Rhodobacter capsulatus, Escherichia coli, and Klebsiella pneumoniae, although it is significantly more sensitive to denaturing conditions. In addition to its catalatic activity StCP catalyzes peroxidatic activity with o-dianisidine, diaminobenzidine, pyrogallol, NADH, and NADPH as electron donors. The enzyme is a tetramer with identical subunits of 61,000 Da molecular weight. StCP shows a typical high-spin ferric heme spectrum with a Soret band at 405 nm and a peak at 632 nm, and binding of cyanide causes a shift of the Soret band to 421 nm, the appearance of a peak at 537 nm, and abolition of the peak at 632 nm. Reduction with dithionite results in a decrease in the intensity of the Soret band and its shift to 436 nm, and in the appearance of a peak at 552 nm. The pH optimum is 6-6.5 and 5.4 for the catalatic and peroxidatic activities, respectively. Fifty percent of the apparent maximal activity is reached at 3.4 mM and 0.26 mM for the catalatic and peroxidatic activities, respectively. The enzyme is inactivated by ethanol/chloroform, and is inhibited by KCN and NaN3, but not by the typical catalase inhibitor 3-amino-1,2,4-triazole.     

The effect of nitrite on cytochrome oxidase

Nitrite inhibits the oxygen uptake by the system ferrocytochrome c-cytochrome oxidase with Ki = 1.5 mM. In the absence of ferrocytochrome c the oxygen uptake by cytochrome oxidase in the presence of nitrite was observed indicating that the enzyme has some nitrite oxidase activity. Nitrite induces changes in optical difference spectra of cytochrome oxidase and, in particular, the formation of the transient band at 607 nm. The reciprocal relation was observed between the intensity of this band and the rate of the oxygen uptake by cytochrome oxidase. This means that the form of the enzyme with this band does not involved in the nitrite oxidase activity. It is suggested that the nitrite oxidase activity relates to the oxygen binding site rather than the cytochrome c binding site of the enzyme.     

Oxidation of sulphide by cytochrome aa3

The effectiveness of H2S as an inhibitor of cytochrome c oxidase increase (Ki decreases) with sulphide concentration. A spectroscopic change in cytochrome aa3 is induced aerobically by sulphide at the same rate as that calculated for inhibition. The initial spectroscopic product is not inhibited, but an 'oxygenated' (oxyferri) form of the enzyme. Stoichiometric sulphide addition to cytochrome aa3 under anaerobic conditions produces another low-spin form of the enzyme; subsequent admission of oxygen gives rise to the 607 nm compound. At high enzyme levels sulphide itself acts as a substrate measured polarographically, with an oxygen uptake proportional to the amount of sulphide added. Binding of sulphide to ferric enzyme probably causes reduction at the oxygen-sensitive a3-Cu centre, which is followed aerobically by reoxidation to the oxyferri state via the 607 nm intermediate. A stable sulphide complex is formed only after the reduction of cytochrome a; but once formed this inhibited species is retained if cytochrome a is reoxidized.     

Perturbation of Pseudomonas cytochrome oxidase by guanidine hydrochloride to detect 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.     

High-pressure investigations of cytochrome P-450 spin and substrate binding equilibria

The effects of high pressure (1-2000 bar) on the spin state and substrate binding equilibria in cytochrome P-450 have been determined. The high-spin (S = 5/2) to low spin (S = 1/2) transition of the ferric hemoprotein was monitored by uv-visible spectroscopy at various substrate concentrations. Increasing hydrostatic pressure on a sample of substrate-bound cytochrome P-450 resulted in a decrease in the high-spin fraction as monitored by a Soret maxima at 391 nm and an increase in the low-spin 417-nm region of the spectrum. These pressure-induced optical changes were totally reversible for all pressures below 800 bar and were found to correspond to simple substrate dissociation from the enzyme. High levels of the normally metabolized substrate, d-camphor, corresponding to a 99.9% saturation of the hemoprotein active site (50 mM Tris-Cl, 100 mM KCl, pH 7.2) completely prevented the pressure-induced high-spin to low-spin transition that is observed at less than saturating substrate concentrations. A gradual increase in the formation of the inactive P-420 form of the cytochrome was noted if the pressure of the sample was increased above 800 bar. These pressure-linked spectral changes were used to determine the microscopic volume change accompanying substrate binding, which was found to be -47.0 +/- 2 ml/mol (pH 7.2) which represents a substantial change for a ligand dissociation reaction. The observed volume change for camphor binding decreases to -30.6 +/- 2 ml/mol at pH 6.0, suggesting the involvement of a linked proton equilibrium. Various substrate analogs of camphor induce varying degrees of low-spin to high-spin shift upon binding to ferric cytochrome P-450 (3). The volume changes for the dissociation of these substrates were very similar to those obtained with camphor. The conformational changes associated with a shift from high- to low-spin ferric iron appear to be small in comparison to the overall macroscopic changes in volume accompanying substrate binding to the enzyme.