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Biochemistry 1991, 30, 840-850
Proton Interactions in the Resting Form of Cytochrome Oxidaset Pavlos G . Papadopoulos, Scott A. Walter, Jianwei Li, and Gary M. Baker* Department of Chemistry, Northern Illinois University, Dekalb, Illinois 601 15 Received June 25, 1990: Revised Manuscript Received October 8, 1990
ABSTRACT: The effect of p H 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 u3 in the resting enzyme. Changing the p H within the range 6.5-10.5 was found to reversibly shift the position of the Soret band over an 1 1-nm range. The lower p H 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. T h e 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 p H 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 pK, of 7.8. Magnetic circular dichroism (MCD) spectra of the low-pH form a t 416 nm, the high-pH form a t 427 nm, and the cyanide derivative a t 428 nm were compared. No evidence of a high-pH-dependent low-spin transition or a change in the redox state of cytochrome u3 was found, confirming earlier work [Baker, G. M., Noguchi, M., & Palmer, G. (1987) J . Biol. Chem. 262, 595-6041, Subtraction of ferricytochrome a [spectrum taken from Vanneste, W. H . (1966) Biochemistry 5 , 838-8481 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. T h e difference extinction change a t 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 a t 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. Two-exponential fitting gave observed rate constants of 2 X lo-* and 3 X s-l for both the low- and high-pH-induced kinetics, with the rapid phase accounting for 4 5 3 5 % of the total absorbance change. Incubation times of 2-3 h were generally required to achieve apparent equilibrium following a 1.6-2.0 unit change in pH. T h e low-pH-induced shift in the Soret band was not kinetically correlated with the fast to slow conversion in cyanide binding behavior that is also known to occur a t low pH (Baker et al., 1987). Various explanations for the pH-induced shift in the absorption profile of cytochrome u3 are considered. A model based on external point charge interaction with the T-T* Soret resonance is consistent with the available data.
C y t o c h r o m e oxidase, a metalloenzyme that functions as the terminal electron acceptor in respiration, catalyzes two important reactions: the reduction of oxygen to water, and the translocation of protons across a lipid membrane (Krab & Wikstrom, 1987; Brunori et al., 1987; Capaldi, 1987). A persistent problem in elucidating the active-site structure and physiological properties of cytochrome oxidase has been the heterogeneity that is often associated with purified preparations of the enzyme. Heterogeneity can involve the entire protein, as evidenced by different aggregation states and variable subunit compositions, or it can reflect the active site, in which one or more redox centers show variable electronic and vibrational structure. Both types of inhomogeneity can complicate kinetic and spectroscopic measurements and possibly reflect nonphysiological forms that arise during the course of purification. In particular, cytochrome a3 in resting enzyme is known to be heterogeneous by several criteria, including multiphasic cyanide binding kinetics, variable amplitude in ‘This investigation was supported by American Heart Association, Illinois Affiliate, Grant C-03 and by Biomedical Research Grant Program Award BRSG SO7 RR07176, Division of Research Resources, National Institutes of Health. *Address correspondence to this author.
0006-296019 110430-840$02.50/0
a low-field electron paramagnetic resonance (EPR) signal designated g’ = 12, and different positions of the optical Soret band. The first two criteria have been extensively documented (Van Buuren et al., 1972; Greenaway et al., 1977; Brudvig et al., 1981; Naqui et al., 1984; Baker et al., 1987), although the structural origins of these effects are not yet understood. In contrast, the lack of constancy in the Soret band of cytochrome oxidase has received little systematic attention. A review of the literature indicates that various purification schemes produce rather different results. Hartzell et al. (1978) reported a Soret maximum of 417 nm for their preparations but noted that other methods showed more red-shifted maxima, by as much as 7 nm. Yoshikawa et al. (1977) and Yu et al. (1975) have reported 419-420 nm for their resting enzyme. Babcock et al. (1976), using a modified version of the Hartzell and Beinert (1974) method, observed a range of maxima in their preparations from 418 to 422 nm. Tiesjema et al. (1972) and Yoshikawa et al. (1988) have reported routine observations of 422-424 nm.
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Abbreviations: CHES, 2-(N-cyclohexylamino)ethanesulfonic acid; HEPES, N-(2-hydroxyethyl)piperazine-N’2-ethanesulfonic acid; DM, n-dodecyl P-o-maltoside; SDS, sodium dodecyl sulfate; EPR, electron paramagnetic resonance; MCD, magnetic circular dichroism.
0 199 1 American Chemical Society
Spectral Effects of pH on Cytochrome Oxidase Although no clear explanation for the different positions of the Soret band has been reported, it presumably stems from the choice of experimental conditions during and after purification. There is precedent for believing that pH is a factor. Kornblatt (1980) observed 3-nm blue shifts in the Soret band when titrating dilute solutions of resting enzyme from pH 7.7 to 6.7. A similar low-pH-induced blue shift was reported by Fabian and Malmstrom (1989) after titration from pH 8.2 to pH values as low as 6.0. The shift in the Soret band consisted of a fast component that was complete within 5 min followed by a slower change, the latter being attributed to enzyme denaturation. Baker et al. (1987) also showed that the Soret band slowly blue-shifted at low pH, from 424 to 419 nm in 10-1 2 h, but that the effect could be reversed by titration back to high pH, a result that is not consistent with denaturation. Various investigators have reported high-pH-induced red shifts in the Soret band of resting enzyme, but, as with the low-pH case, there is controversy over the origin of this effect. The red shift observed by Callahan and Babcock (1983) in the pH range 8.5-10.2 was interpreted, based on optical and resonance Raman data, to reflect a high- to low-spin transition in cytochrome a3, consistent with the high-pH behavior observed for various other native high-spin ferric heme proteins such as metmyoglobin and cytochrome c peroxidase (Morishima et al., 1977; Dhaliwal & Erman, 1985). Using magnetic circular dichroism (MCD), Baker and Palmer (1987) reexamined the high-pH effect but were unable to find evidence of a low-spin transition. The MCD had nonetheless established that the Soret shift was due to an effect of pH on cytochrome a3,and not on cytochrome a . Schoonover et al. (1988) compared the resonance Raman properties of two forms of resting enzyme having Soret maxima at 417 and 424 nm (obtained by low- and high-pH incubation, respectively) and further showed that the high pH-induced red shift in the Soret band could not be related to a change in heme redox state or coordination number. A number of alternative explanations for the pH-dependent shift were considered. The current study attempts to clearly document the effect of nondenaturing pH on the near-UV absorption spectrum of resting cytochrome oxidase. The results focus on three important objectives: (1) to demonstrate that pH-induced optical changes can be used as a probe of active-site heterogeneity: (2) to discuss the relationship of these changes to the fast and slow cyanide binding forms; and (3) to provide a working explanation for the pH effects on the Soret band of resting enzyme. The last objective draws on the current investigation as well as on previously published work. MATERIALS AND METHODS Purification of Cytochrome Oxidase. Enzyme was isolated from bovine heart as described by Baker et al. (1987). The approach incorporates a series of minor changes to the original method of Hartzell and Beinert (1974). A pH of 8.8-9.0 (2-6 "C), somewhat higher than that reported previously, was maintained and monitored with a pH electrode during the cholate/ammonium sulfate fractionation stages. The final precipitate of enzyme was dissolved in 0.1 M C H E S K O H ' buffer (pH 8.8, 4 "C) with 0.1% (w/v) n-dodecyl 0-Dmaltoside (DM). Starting with 3 kg of heart mince generally gave about 200 mg of purified enzyme. Titration Conditions. For most experiments, aliquots of stock enzyme were diluted into either 50 mM CHES. KOH/O. 1% (w/v) DM or 0.1 M K2HP04.KOH/0. 1% (w/v) DM buffers to give 5-10 pM heme a at pH 8.8 (20 "C). Titration to lower pH values was accomplished with either 2.5
Biochemistry, Vol. 30, No. 3, 1991 841 M HEPES/O.l% (w/v) DM, pH 5.3 (HEPES titrant), or 1.25 M KH2P04in 1 N H,PO,/O.l% (w/v) DM, pH 2.6,20 OC (low-pH phosphate titrant). Higher pH transitions used 1.25 M K,HP04 in 1 M KOH (or 1.25 M K3P04)/0.1% (w/v) DM, pH 12.0-12.3, 20 OC (high-pH phosphate titrant). Dilution factors due to added titrant were 1.02-1.06. Titrant was generally added over a 2-3-min period with stirring, unless otherwise indicated. Potassium sulfate (167 mM; p = 0.5 M) was often included in both buffers and titrants to promote optical clarity at pH values below 7.0 (see Results). Comparisons with different buffers and titrants (fK,S04) demonstrated that the pH-dependent optical shifts were not due to specific buffer or ionic strength effects, as described under Results. All experiments were conducted at 18-20 "C unless otherwise indicated. The pH was measured with an Orion EA 920 ion analyzer equipped with automatic temperature compensation probe and a glass combination 3.5-mm-diameter electrode. Electrode calibration was always performed at the same temperature as the experiment using two buffers with standard values that bracketed the pH of interest. Calibration was checked at several points during an experiment to minimize errors in pH determination. Drifts of no more than 0.08 pH unit at 20 "C were observed during several hours of incubation at any selected pH between 6.3 and 10.5. Enzyme Characterization. Heme a concentration was determined by applying a millimolar extinction coefficient of 20.9 mM-'.cm-' [averaged from Yonetani (1960) and Brunori et al. (1979)l to the dithionite-reduced a-band at 604 nm. Enzymatic activity was assayed and expressed according to the method of Yoshikawa et al. (1977). Instrumentation and Data Analysis. Double-beam and dual-wavelength scans were recorded with a Shimadzu UV-3000 near-UV/visible spectrophotometer equipped with a constant-temperature circulator and a magnetic stirrer. Data acquisition was accomplished through GP-IB interfacing to an IBM AT-compatible computer. Wavelength scan resolution was f0.2 nm at 50 nm/min. Fixed-wavelength assays were programmed to acquire data points every 1 or 2 s. Path length was always 1 cm. Spectral processing and graphics displays were accomplished with Lab Calc augmented with an arithmetic applications package (Galactic Industries Corp., Salem, NH). Presentation quality scans were generated at 300 dpi on a NEC Silentwriter LC-860+ laser printer. All absorbance scans represent "raw" data with no smoothing or offset corrections. Magnetic circular dichroism (MCD) spectra were recorded in a 1-cm cell using a custom-built spectrometer equipped with a superconducting magnet operating at 7 T, as described by Mason (1982). The CD contribution, recorded in the absence of the magnetic field, was subtracted from the spectrum obtained with the field on to give the MCD contribution. The result was expressed as At/T (M-'.cm-'/T), the difference extinction between left and right circularly polarized light on a per mole of heme a basis and normalized to 1 T. The C D spectra were scaled to a (1R)-camphorsulfonic acid standard at 290.5 nm (Schippers & Dekkers, 1981), and the magnetic field was calibrated with ferricyanide, as described by Vickery et al. (1976). Data points were collected at the rate of 100/nm at 10 nm/min using a 3-s time constant and a spectral bandwidth of < O S nm. Spectra were processed with a moving window smoothing routine (Savitzky & Golay, 1964) for display.
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Wavelength (nm) FIGURE 1 : Soret absorption spectra of resting cytochrome oxidase incubated at low pH, at high pH, and with cyanide. All enzyme samples were at 5 p M heme a and were prepared at p H 8.8 in 0.1 M KH2P04.KOH/167 m M K2S04/0.1%(w/v) DM. Treatment of samples with titrant or cyanide occurred after 2 h of incubation at 20 "C. (-) Resting enzyme was titrated to p H 6.7 using low-pH phosphate titrant (see Materials and Methods). The spectrum shown (Soret maximum, 416.4 nm) was recorded after 2 h of incubation at 20 OC. (---) Titration occurred to pH 10.5using high-pH phosphate titrant (see Materials and Methods). After 3 h of incubation, a spectrum with a Soret maximum at 427.0 nm was recorded. Sodium cyanide was added to resting enzyme to give a final concentration of 50 mM CN-, as described under Materials and Methods. The reaction, which displayed a single, fast binding phase, was complete within several minutes, at which time a spectrum was recorded. (-e)
Kinetics. Cyanide binding to oxidized enzyme was initiated by transferring an aliquot from a 4 M stock of NaCNmHCI, pH 8.8, into 2.5 mL of a 5-10 pM heme a solution to give a final concentration of 25-50 mM. Reaction occurred at either pH 8.8 or pH 6.7, as indicated in the appropriate figure legends. The binding was measured as an increase in absorbance at 428 nm. The time course associated with the pH-induced shift in the Soret band was measured by the dual-wavelength method. The absorbance change at 416 nm, relative to that at the isosbestic point, was measured in response to a pH transition. The amount of titrant needed to achieve a given pH change was determined in separate control experiments to allow a single, rapid addition. An enzyme solution at pH 8.7-8.8 was introduced into the spectrophotometer cuvette (1 cm path, 10 pM heme a); the initial time point was recorded, at which time the titrant was added (while stirring) to bring the pH to 6.7-6.8 (total lapsed time between the first and second data points was 8 s). A pH reversal was accomplished by adding enough titrant to the low-pH solution to bring the pH back to 8.8. Dilution factors did not exceed 1.02-1.06X. Specific buffer and titrant compositions are given in the appropriate figure legends.
RESULTS Effect of p H on the Near-UV Absorption Spectrum of Resting Enzyme. Dilution of stock enzyme into pH 8.8 buffer (5-10 pM heme a, 20 "C) showed an initial Soret band position at 422-423 nm. A slight red shift to 423-424 nm occurred during 2 h of incubation, with longer times causing no further changes in the position, intensity, or shape of the Soret band. Titration of 2-h-incubated enzyme to pH 6.7 (20 " C ) resulted in a slow blue shift of the Soret band to 416.4 nm (solid trace, Figure 1). The shift, which occurred over 2 h, was characterized by an increase in extinction and the
progressive appearance of a prominent shoulder on the lowenergy side of the Soret maximum. Continued titration to pH 6.3 caused no further changes in the spectrum.* Replications with different preparations of enzyme indicate 416 nm to be the average limiting position of the Soret band at low pH (the most blue-shifted case was 415.4 nm). Enzyme denaturation is not expected within this low-pH range since the optimal pH for enzyme activity is 6 (Maurel et al., 1978; Kornblatt, 1980). If the pH 8.8 solution (Soret maximum, 423-424 nm) was titrated to pH 10.5, rather than 6.7, the Soret band shifted further to the red, stabilizing at 427.0 nm after 3 h of incubation (dashed line, Figure 1). The shift was associated with a loss of intensity and the development of a prominent, broad shoulder on the high-energy side of the Soret maximum. The addition of cyanide to the pH 8.8 solution caused a similar shift to 428 nm, but the shape and amplitude were different (dotted spectrum, Figure 1). Cyanide caused both an intensification and a narrowing of the Soret band, the bandwidth at half-height being 35 nm for the cyanide-treated case and 45 nm for the high-pH-incubated resting case. Raising the pH of resting enzyme above 10.5 and incubating for longer times resulted in a further loss of Soret amplitude, and at pH values greater than 12, the loss was associated with a blue shift, indicating denaturation. Prolonged incubation of resting enzyme at pH values higher than 10.5 is known to be associated with partial heme denaturation (Baker & Palmer, 1987). Degradation of heme was avoided by maintaining the pH at 10.5 (20 " C )for no longer than 2-3 h. Accordingly, the solid and dashed spectra shown in Figure 1, with limiting Soret band positions at 416 and 427 nm, represent native heme a states for two reasons: (1) The shift response to either pH 6.7 or pH 10.5 was reversible. Both the intensity and position of the Soret band were fully recovered by titration back to pH 8.8. (Evidence of reversibility will be shown in Figure 7.) (2) The pH-induced shift to 427 nm was associated with an isosbestic point (Figure 2), a result that is not consistent with progressive, alkaline-dependent denaturation. Figure 2A shows a series of optical spectra obtained every 20 min after performing a transition to pH 10.1, while Figure 2B reflects a similar series of spectra recorded after a transition to pH 7.4. An arrow in each case indicates the direction of the shift. Each set of spectra shows a clear isosbestic point, establishing the presence of only two absorbing forms at this wavelength. The isosbestic points typically settle in the range 421-427 nm, with the longer wavelength values being typical of high-pH, rather than low-pH, transitions. The data, however, do not eliminate the possibility of additional absorbing species either if they are pH-insensitive or if their pH-dependent absorbance contribution at the isosbestic wavelength is minimal. Nonabsorbing intermediates are also possible since only one isosbestic point is clearly defined in both blue- and red-shifting cases. The most red-shifted case in Figure 2A, obtained after 1.3 h of incubation at pH 10.1, is at 426.9 nm. The retention of the isosbestic point indicates that the red-shifted forms and, in particular, the 427-nm form shown in Figure 1, were not due to denaturation. Additionally, the pattern shown in Figure Below pH 6.7, we occasionally observed turbidity that worsened as the pH was further decreased. Turbidity distorts the spectral profile and leads to artifactual shifts. Optical clarity down to pH 6.3 could be achieved by including 167 mM potassium sulfate (ionic strength, 0.5 M) in the buffer/detergent solution. Below this pH, cloudiness developed. Filtration at this point resulted only in transient clarification, with the turbidity returning after a few minutes. Ionic strength effects on the pH dependence of the Soret band position are currently being examined. On a qualitative level, the data are unchanged.
Spectral Effects of pH on Cytochrome Oxidase
Biochemistry, Vol. 30, No. 3, 1991 843 40
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Magnetic circular dichroism (MCD) spectra of resting enzyme incubated at low pH, at high pH, and with cyanide. Solid, dashed, and dotted spectra correspond to the absorption spectra shown in Figure I . The samples were identical. Instrument settings and scan conditions are described under Materials and Methods. FIGURE 3:
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Wavelength (nm) Time-dependent response of the Soret band to a change in pH. Heme a concentration was 5.1-5.3 gM in all cases. Optical spectra were recorded every 20-30 min following the transition to low or high pH. (A) Stock enzyme was diluted into pH 10.1 buffer containing 50 mM CHES-KOH, 167 mM K2S04,and 0.1% (w/v) DM. The first recorded spectrum showed a Soret maximum (Ama,) at 424.5 nm (obtained after 20 min) which shifted to 426.9 nm after 1.3 h of incubation at 20 OC. The isosbestic point (Aim) was at 425.0 nm. (B) Stock enzyme was diluted into buffer at pH 10.1 containing 50 mM CHES-KOH and 0.1% (w/v) DM. After 3.8 h of incubation at 18 OC, enough HEPES titrant (see Materials and Methods) was added to lower the pH to 7.4. The A,, just prior to the low-pH transition was 424.4 nm. The first spectrum was recorded after IO min at pH 7.4 and showed a A,, at 422.5 nm. The band continued to shift during 2.3 h of incubation at 18 OC until it reached a stable value at 417.5 nm. The isosbestic point was at 424.3 nm. Spectra in (A) and (B) were from different enzyme preparations. FIGURE 2:
2A could be reversed by cycling the pH back to 8.8 (see Figure 7). The effect of a pH transition to 7.4 is shown in Figure 2B. The Soret band was observed to blue shift until it reached a stable position at 417.5 nm. The near-UV absorption band retained its shape and intensity with activities that remained constant at 1 .O-1.4 s-'-(nmol of heme a)-l over a 24-h period at room temperature. In contrast, the pattern shown in Figure 2A required that the time of incubation at pH >10.0 be kept to a minimum. For example, more than 3 h of incubation at pH 10.5 led to irreversible optical changes such as a loss of the isosbestic point due to a gradual attenuation of the Soret band intensity, a result that probably reflects denaturation. A comparison of the activities of the blue- and red-shifted forms in Figure 1 showed slightly different amounts of recoverable activity after 2 h of incubation, with the low-pH case being about 20% higher [1.0-1.4 s-l.(nmol of heme a)-'] compared with values of 0.8-1 .O s-l.(nmol of heme a)-' for the high-pH case, each assayed at pH 6.0. The slight decrease in activity over this time period probably reflects denaturation of Cu,, as judged by a small, irreversible attenuation of both the 830-nm band and the g = 2 EPR intensity (Baker & Palmer, 1987). Deviation from the isosbestic point after ex-
cessive incubation at pH 10.5 was accompanied by further loss of activity, decreasing from about 1 to 0.2-0.4 s-'.(nmol of heme a)-I over a 20-h period at room temperature. Severe denaturation at higher pH (> 13) resulted in a general loss of extinction and a severely broadened and blue-shifted absorption maximum at about 406 nm. Effect of p H on the Near-UV MCD Spectrum of Resting Enzyme. Several reports have suggested that alkaline pH induces a transition to low spin in cytochrome a3 (Lanne et al., 1979; Callahan & Babcock, 1983). Baker et al. (1987) reexamined this question using magnetic circular dichroism (MCD) and magnetic susceptibility as probes. Although no evidence of a low-spin transition was found, two forms of resting enzyme were compared that had Soret maxima at 417 and 423 nm. The current study examines the MCD spectra of the more widely separated forms at 416 and 427 nm shown in Figure 1. The Soret MCD of the blue- and red-shifted extremes provides a more critical test of two important questions: (1) Is the pH effect confined to cytochrome a3,and (2) has there been a transition to low spin induced by high pH? Babcock et al. (1 976) showed that the Soret MCD of resting enzyme was due entirely to cytochrome a, the derivativeshaped C term being characteristic of low-spin ferric heme. Additional evidence for this came from earlier analyses of photochemical action spectra of CO-ligated oxidase in which Vanneste (1966) had determined the absorption maximum of cytochrome a to be at 426 nm, a value that corresponded very closely with the MCD zero crossover point at 427 nm. Our rationale was that if the pH-dependent 1 1-nm band shift was due to an effect on cytochrome a, then the crossover point of the MCD spectrum should shift accordingly. Failure to observe a shift would be consistent with an effect on cytochrome u3,which is not MCD-detectable in its high-spin state. The solid and dashed traces in Figure 3 compare the MCD spectra of the blue- and red-shifted forms having Soret maxima at 416 and 427 nm, corresponding to the absorption spectra in Figure 1. The MCD crossover points are identical, with both at 426.2 nm. The pH-dependent 1 1-nm band difference is therefore due to a change in the electronic structure of cytochrome a3, in accord with the findings of Baker et al. (1987). Cyanide binding to cytochrome oxidase causes a quantitative low-spin conversion and a correspondingshift in the Soret band
844 Biochemistry, Vol. 30, No. 3, 1991 to 428 nm. The similar Soret band position of the alkaline derivative of resting enzyme (dashed spectrum, Figure 1) suggested that it was also low spin. The Soret MCD provides a convenient quantitative measure of low-spin content. Vickery et al. (1976) compared the MCD of a variety of metmyoglobin/ligand complexes which were determined by magnetic susceptibility to have different low-spin proportions. An approximate linear correlation was found between the MCD trough intensity and the fraction of low-spin iron present. Babcock et al. (1976) also showed that the reaction of resting cytochrome oxidase with cyanide resulted in a 2-fold increase in the trough amplitude of the MCD peak at 434 nm, a result that was consistent with a low spin transition in cytochrome a3. Figure 3 compares the MCD amplitudes of the high- and low-pH forms with that of the cyanide-bound derivative of resting e n ~ y m e . The ~ blue-shifted form at low pH is expected to be high spin and to therefore have an MCD amplitude that reflects only cytochrome a . In accord with the observation of Babcock et al. (1 976), reaction with cyanide was found to increase the trough amplitude at 434 nm from -22.7 (solid trace) to -44.4 Ae/T (dotted trace). In contrast, the MCD of the high-pH case (dashed trace) showed a small decrease at 434 nm rather than the 2-fold increase that would be expected if cytochrome a3 was low spin and ferromagnetically coupled to Cu,. [The possibility of a diamagnetic ground state due to antiferromagnetic coupling with CuB is considered unlikely based on the observed retention of the high-spin charge-transfer band in the visible MCD spectrum (Baker & Palmer, 1987).] The red shift to 427 nm is therefore not the result of an alkaline-dependentlow-spin transition, and explains why the near-UV absorption spectra of the alkaline and cyanide derivatives are different in both shape and intensity (dashed and dotted spectra, Figure 1). The MCD results also indicate that the two absorbing forms that give rise to the isosbestic point in Figure 2 reflect different pH-sensitive states of cytochrome a3, and not cytochrome a. Nature of the Two Forms of Cytochrome a3. The highpH-induced red shift to 427 nm indicates that cytochromes a and a3 absorb maximally at similar wavelengths. Yet, despite the increased overlap in absorption profiles, there is a marked loss of intensity that cannot be attributed to denaturation. In an effort to better visualize the two cytochrome a3 bands that give rise to the isosbestic point, we subtracted the contribution of cytochrome a from a series of red- and blue-shifting spectra similar to those shown in Figure 2A,B. The spectrum reported by Vanneste (1 966) for ferricytochrome a was used to perform the subtraction after scaling to the correct concentration, an approach that is valid since the MCD data indicate that cytochrome a remains largely unchanged over the working pH range. Figure 4A shows the result of this subtraction for a blue-shifting series, in this case from 422.5 to 417.5 nm. The actual spectrum for cytochrome a that was used to perform the subtraction is shown in the inset. The spectrum was extrapolated below 400 nm using Gaussian curve fitting (see figure legend). The vertical dashed lines in Figure 4A indicate the approximate positions of the two absorption bands that give rise to the isosbestic point. The peak at 413 nm intensifies at the expense of the other transition at 430 nm. The ab-
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Purified stock enzyme, upon dilution into pH 9.0 buffer, shows a Soret maximum at approximately 423 nm. The cyanide binding reaction is complete in about 2 min at 25 mM cyanide and shows a single-exponential binding phase with an observed rate constant of 0.04 s-l, in agreement with Baker et al. (1987). A much slower reacting form of resting enzyme is evident only after low-pH incubation, with hours now required for complete reaction rather than minutes.
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Wavelength (nm) FIGURE 4: Subtraction of ferricytochrome a from a series of blueand red-shifting Soret band spectra. Heme a concentration was 4.9 pM. (A) Buffer and titrant conditions were as in Figure 2B. (B) Buffer and titrant conditions were as in Figure 2A. Vertical arrows in (A) and (B) indicate the direction of amplitude change associated with two peaks with center frequencies denoted by dashed, vertical lines. The inset in (A) shows the spectrum of cytochrome a, normalized to 4.9 pM total heme a, that was used to perform the subtraction. Two Gaussians with center frequencies at 426 and 390 nm were used to iteratively generate the spectrum for cytochrome a reported by Vanneste (1966) and to extrapolate the high-energy descent from 400 to 380 nm.
sorption envelope for cytochrome a3 narrows and becomes more symmetric as the Soret maximum shifts to higher energy, showing only a minor contribution from the 430-nm band for the most blue-shifted case. Comparison of this blue-shifted spectrum with Vanneste’s (1966) spectrum for cytochrome a3 shows good quantitative agreement. Vanneste reported an absorption maximum at 414 nm, compared with our result of 413 nm; the widths at half-height were 33.5 nm (Vanneste) and 35 nm, and the amplitudes at A, (after correcting for the difference in heme a concentration) were 0.166 A in Vanneste’s case and 0.169 A in our case. The subtraction of cytochrome a was also performed for a series of red-shifting spectra, with the result shown in Figure 4B. The Soret band in this case shifted from 422.3 to 426.3 nm in response to a high-pH transition. The a3 band on the high-energy side of the isosbestic point showed a maximum at 414 nm, which is slightly red-shifted compared with that in Figure 4A. Several comparisons of red- and blue-shifting cases have shown that the position of this high-energy band, after subtraction, can vary from 413 to 416 nm. Regardless of the actual position, Figure 4B shows that the high-pH transition causes a progressive loss in amplitude at 414 nm, while the transition at 430 nm becomes more prominent. Since the observed position for the Soret band at 426.3 nm is close to the red-shifted limit at 427 nm (from Figure l ) , a simple interpretation predicts that the absorption peak at
Spectral Effects of pH on Cytochrome Oxidase
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I pk?=
*
414 nm would have decreased to nearly zero amplitude. Similarly, the blue-shifted extreme predicts a complete loss of the 430-nm transition. Although the band a t 430 nm behaves as expected (Figure 4A), the situation at 414 nm is not as clear since a large background absorption is still evident for the red-shifted limit (Figure 4B). However, if the highpH-dependent loss of amplitude is followed a t 414 nm, it is observed that the peak position progressively shifts to about 41 0 nm, demonstrating the contribution of another high-energy band (this trend is consistently evident for different preparations of enzyme). This background absorption is too large to be regarded as an artifact of the subtraction technique. Additionally, it is not pH-sensitive and is largely eliminated by treatment with cyanide, identifying it as a cytochrome u3,or possibly Cu,, transition. The loss of this band after cyanide binding can be seen in Figure 1 by comparing the bandwidths a t half-height of the cyanide- and alkaline-treated cases. Bandwidths of 35 and 45 nm, respectively, are evident, as discussed previously. Although both cases have Soret maxima a t 427-428 nm, it is clear that the high-pH case is broader because of a prominent high-energy ~ h o u l d e r . ~ Figure 5 shows how the difference extinction coefficients a t 414 and 430 nm change as a function of the observed position of the Soret band. When the Soret band is a t 427 nm (at pH 10.5), the 414-nm band area is assumed to be zero, with all of cytochrome u3 in the 430-nm form. Similar reasoning is applied to a low-pH transition, when the Soret band is at 416 nm (at pH 6.7). All of the cytochrome a3 is presumed to be in the 414-nm form. Four different preparations of enzyme are represented using difference extinction data calThe alkaline resting form, the hydrogen peroxide adduct, and the cyanide adduct all have Soret maxima at 427-428 nm. The alkaline- and peroxide-treated cases exhibit a high-energy shoulder, but the cyanide derivative does not. Interestingly, the peroxide adduct also shows no increase in MCD amplitude (Carter et al., 1981), much like the alkaline-treated case. Detailed comparisons of the pH sensitivity of each form are currently being investigated.
4161
7.8
0
I 6
845 I
I
418 420 422 424 426 Soret Maximum (nm)
FIGURE 5: Change in difference extinction coefficients at 414 and 430 nm following a pH transition. Several series of spectra from four preparations of enzyme were obtained using the various buffer and titrant conditions described in Figures 1 and 2. Difference extinction data were extracted from red- and blue-shifting spectra (open and closed symbols, respectively),including those shown in Figure 2A,B. The squares (0, u) denote the difference extinction coefficients at 414 nm whereas the circles (0, 0 ) depict the corresponding values at 430 nm. The coefficients were calculated relative to assumed zero values (see text) using heme a concentrations determined according to Materials and Methods. Linear least-squares fits are shown for each set of A€ data at 414 and 430 nm. The slopes, respectively, are -2.2 0.3 and 0.6 0.1 mM-l cm-l/nm based on 99% confidence boundaries. The absolute extinction coefficient at the Soret maximum varies linearly from 81.9 mM-I.cm-l at 415.7nm to 70.5 mM-'.cm-' at 427.0nm.
*
Biochemistry, Vol. 30, No. 3, 1991
7
I
I
I
I
0
9
10
11
PH
Titration profile of the Soret band maximum. Data shown reflect the pH titrations of nine different preparationsof resting enzyme using the approach described under Materials and Methods. The phosphate buffer/titrant system was used for most of the titrations. The different band positions at any given pH depend on preparation variability and not on the choice of buffer or titrant (see text). The apparent equilibrium value for the Soret band position, following a transition to either low or high pH, was reported only after obtaining two or three identical spectra recorded 10 min apart. Equilibration time at each pH value was generally 2-3 h at 18-20 OC. The solid = [LI + curve represents a least-squares fit to the equation A, L2(10pH-pK8)]/(10pH-pKa+ l), where L1 and L2 are the lower and upper limits of the titration profile, respectively. None of the parameters were fixed. FIGURE 6:
culated from different sets of blue- and red-shifting spectra. A comparison of the slopes for the linear least-squares fits of the two scatter profiles indicates that the band at 430 nm has a transition probability that is about 4-fold lower than the band a t 414 nm (assuming that the amplitude is proportional to the area under the transition). A linear dependence a t all wavelengths is expected if changes in amplitude are simply related to concentration differences between two absorbing species, as implied by the occurrence of an isosbestic point. Heme u concentrations in all cases were determined by using an extinction coefficient of 20.9 mM-km-' at 604 nm for the reduced a band (see Materials and Methods). Most of the change a t this wavelength (about 80%) is due to reduction of cytochrome a (Vanneste, 1966; Carter & Palmer, 1982) and is therefore expected to be relatively insensitive to variations in pH. This approach also gave the extinction coefficient scale that is shown in Figure 1, with results for the blue-shifted band that are in reasonable agreement with published results (8 1.2 mM-'.cm-' for,,A a t 416.4 nm). A comparison of the blueand red-shifted spectra in Figure 1 (solid and dashed spectra) shows a millimolar difference extinction coefficient a t 414 nm of 23.5. Reference to Figure 5 indicates this to be the approximate value that is expected when the Soret band is observed a t 416.4 nm. p H Titration Profile of the Soret Band. Figure 6 addresses the question of whether the Soret band can be titrated to different apparent equilibrium values that depend on pH. Results from nine different enzyme preparations are shown in Figure 6 in order to give some estimate of scatter. Values for the Soret maximum were obtained by titrating a dilute solution of resting enzyme (5-10 p M heme a ) to the desired pH and then recording a series of optical spectra, as in Figure 2, until no further shift in the Soret band position was observed. The results were not affected by a variety of experimental approaches. For example, either a given experiment relied on sequential titration of the same enzyme solution to give different p H values, waiting long enough a t each p H until the
Papadopoulos et al.
846 Biochemistry, Vol. 30, No. 3, 1991 Soret band had stabilized, or it involved addition of titrant to several solutions of enzyme that incubated simultaneously, thereby eliminating the "aging" variable that characterizes the first approach. The first approach, however, has the advantage of allowing the monitoring of an isosbestic point during the incubation period. Within the observed scatter, there was also no indication that changes in ionic strength altered the titration profile, since some of the data p i n t s in Figure 6 were obtained in the absence of potassium sulfate2 and included only CHES/HEPES as the buffer and titrant. A nonlinear least-squares fit of the titration data, assuming a single acid/base group (see figure legend), gave an apparent pK, of 7.8.5 The limiting positions defined by the fit (416.6 and 424.4 nm) are different than the 41 6- and 427-nm limits shown in Figure I . The discrepancy stems from the scatter that is evident in a large number of comparisons with different enzyme preparations. For example, similar incubations of nine different preparations a t p H 10.0 have shown apparent equilibrium band positions ranging from 423.5 to 427.0 nm. The experimental origin of this variability, which may be thought of as a type of variable pH resistance, is not yet clear, but it may be related to different extents of subunit 111 removal during enzyme purification (work in progress). Kinetics of the pH-Induced Shift in the Soret Band Position. Figure 2 showed that a pH transition led to a shift in the Soret band of cytochrome oxidase that generally required 2-3 h to reach a stable, apparent equilibrium position. In the course of these studies, it was observed that rapid additions of titrant (2-8-s mixing times) were always associated with a 2-3-nm shift before the first optical spectrum could be recorded, indicating the presence of a fast component. To resolve this component, it was necessary to do a fixed-wavelength assay. Figure 5 showed that the position of the Soret band was linearly related to the change in absorbance a t 414 and 430 nm. The time course of the shift can therefore be followed by measuring the change in absorbance a t a fixed wavelength, although the high-energy side is preferred since the absorbance changes are larger. The dual-wavelength method was used to generate the time scans in Figure 7A in order to cancel the dilution effect of added titrant that would otherwise interfere, as in a conventional double-beam experiment, with the observation of a fast component. The reference wavelength was set to the isosbestic point, which was determined and averaged from several replicate experiments of the type shown in Figure 2. The two time courses in Figure 7A represent a pH reversal experiment, in which enzyme was first titrated from pH 8.7 to 6.8 followed by titration back to pH 8.7, as described under Materials and Methods. A rapid increase in absorbance (AA416-421.0nm),followed by a slower exponential change, was recorded in response to the low-pH transition. About 20% of the fast phase occurred within the 8-s mixing time of the experiment. By use of two-exponential curve fitting, the rate constant for the fast component, which accounted for 53% of the total absorbance change, was estimated a t 2 X s-l, while that of the slow phase was determined to be 3 X s-I. The initial position of the Soret band, just prior to the addition of titrant, was 421.6 nm1.6 At the end of the time More recent work shows that incubation times of 16-17 h at pH 8.6, 20 OC, can lead to Soret maxima that are slightly blue-shifted, 1-2 nm, from the corresponding values shown at pH 8.6 in Figure 6. The data shown in this figure were all obtained after a 2-3 h period of incubation. Slight additional blue-shifting after much longer incubation times suggests that the true pK, may be greater than 7.8. (S. A. Walter, P. G. Papadopoulos, J. Li, and G. M. Baker, unpublished results).
.06
A
9
f
.010-.01 -.02
- h,,
.,03
50
0
e,
,61Ai,
h,,=
= 421.6 nm
422.9 nm 150
100
Minutes
= 421.0 nm
B/
,8
1 hi,=
425.6 nm
--
390
410
430
450
390
410
430
450
Wavelength (nm) FIGURE 7: Kinetics and reversibility of the pH-induced Soret band shift. Resting enzyme at pH 8.7 and I O fiM heme a was prepared in buffer containing 50 mM K2HP04.KOH, 167 mM KzSO4, and 0.1% (w/v) DM. Time scans were recorded by using dual-wavelength spectrophotometry,as described under Materials and Methods. (A) Resting enzyme incubated at pH 8.7 for 10 min was rapidly titrated to pH 6.7 using low-pH phosphate titrant (see Materials and Methods). An increase in absorbance at 416 - Xiso was followed over a 2.8-h period, at which time high-pH phosphate titrant (as defined under Materials and Methods) was quickly added to bring the pH back to 8.7. The time course associated with a decrease in absorbanceat 416 - hisowas again measured over 2.8 h. Soret maxima just before the titrations and after 2.8 h following each pH transition are indicated in the figure. Observed rate constantsextracted from a tweexponential and 3 X IO4 s-I for both least-squares fit of the data were 2 X the low-pH- and high-pH-induced cases. (B) A control spectrum at pH 8.9 (Soret maximum, 421.9 nm) and three spectra obtained at 10, 30, and 180 min after titration to pH 6.7 are shown. Titration conditions were as described in (A). The Soret band blue-shifted at low pH until it reached a stable value at 417.1 nm after 3 h of incubation. The isosbestic point at 421.0 nm was used as the reference wavelength for the low-pH-induced kinetic measurement shown in (A). (C) The sample at 417.1 nm in (B) was titrated back to pH 8.9 by rapid addition of high-pH phosphate titrant. Spectra were recorded at 10, 30, and 180 min following the transition to pH 8.9. The Soret band shifted during this 3-h period from 417.1 to 422.7 nm and showed an isosbestic point at 425.6 nm. The reference wavelength for the high-pH-induced kinetic measurement shown in (A) was reset to this value. This caused a ositive shift in the to 0.057 dual-wavelength absorbance, from 0.01 5 (A,486~"-"z1~0"m) (AA4'6.w425.6"m). The most blue-shifted spectrum in (B), obtained at pH 6.7, is included in (C) for comparison. All spectra shown in (B) and (C) are normalized to the same heme a concentration.
scan, the band had reached an apparent equilibrium value of 417.2 nm. Since the absorbance change associated with a shift from 427 to 416 nm (cf. Figure 5) is assumed to reflect highto low-pH conversion in 100% of the (1, centers, then, by proportion, the shift from 421.6 to 417.2 nm observed in Figure 7A must reflect a change in about 40% of the a, centers. Figure 7B shows the results of a separate experiment in A Soret maximum of 421.6 nm did not reflect a stable equilibrium value since the transition to low pH occurred within IO min after preparation of the enzyme solution in pH 8.8 buffer. Longer initial incubation times are not used in these kinetic experiments since it becomes more difficult to achieve an isosbestic point during a pH reversal. The pH reversal shown in Figure 7 A records the actual approach to an apparent equilibrium value of 422.9 nm, which is more typical of pH 8.8 (compare Figure 6).
Spectral Effects of pH on Cytochrome Oxidase which the spectrum at pH 8.7 is compared to three spectra obtained at different times after the transition to pH 6.8. Correcting for the dilution factor due to added titrant leads to spectra that show a clear isosbestic point in all four cases. Since an isosbestic point implies a binary system, a low-pH transition should simply induce the conversion to a new equilibrium state, a process that is expected to show a single-exponential phase rather than the biphasic response shown in Figure 7A. The same figure also shows that the pH response was reversible. After completion of the low-pH time scan (approximately 3 h), the reference wavelength was reset (see figure legend) to reflect the isosbestic point obtained for a series of red-shifting spectra in a separate control experiment. The enzyme solution (Soret maximum, 417.2 nm) was then titrated back to pH 8.7, and the loss of absorbance associated with the red shift was recorded. As with the low-pH transition, there was a biphasic change in absorbance with the fast component accounting for 45% of the total absorbance change. The rate constants for the fast and slow phases were identical with those observed for the low-pH-induced kinetics. At the end of the 3-h incubation at pH 8.7, the Soret band had shifted to 422.9 nm, a value that is close to the expected apparent equilibrium position (cf. Figure 6). The number of a3 centers affected by the high-pH transition was estimated to be 52%, using data from Figure 5 . The discrepancy between this percentage and the 40% observed for the transition to pH 6.7 stems from the initial use of enzyme that had not fully equilibrated at pH 8.8 prior to the transition to low P H . ~ Figure 7C represents the series of spectra obtained after an identical pH reversal. The most blue-shifted spectrum in Figure 7B is reproduced in Figure 7C since it represents the control spectrum just prior to the addition of high-pH titrant. The remaining three spectra were recorded over a 3-h period following the transition back to pH 8.8. An isosbestic point was again evident for the series of spectra, including the control. (The origin of the red shift in the isosbestic point following a high-pH transition is not clear, but it is highly reproducible.) The pH reversibility and the clear isosbestic points shown in Figure 7B,C eliminate the possibility of heme denaturation during the slow shift in the Soret band, contrary to the interpretation of Fabian and Malstrom (1989). Transitions to pH values greater than 8.8 introduce complications. For example, at pH 10.5, there is a rapid, reversible attenuation in the Soret band amplitude that causes the lowpH control spectrum to deviate from the isosbestic point (data not shown), implying a pH-dependent change in a third absorbing species. The anomalous loss of Soret intensity correlated with the slight decrease in MCD amplitude shown for the high-pH-treated case in Figure 3, implicating the third species as cytochrome a. The effect may be related to the gz = 2.6 EPR signal. The intensity of this signal, which is due to cytochrome a (Baker & Palmer, 1987), is not observed until the pH is raised above 9, and at pH 10.5-1 1.0, it can account for 25-30% of the total low-spin content.' The lower pH of
' A t high pH, a new low-spin signal appears in the EPR spectrum of resting enzyme (Baker & Palmer, 1987). The new signal, which has a g, value at 2.6, becomes more intense as the pH is raised above 9. At pH values less than 9, the signal disappears, and its intensity is recovered in the g, = 3.0 signal, thus identifying it as originating from cytochrome a. In contrast, Hartzell and Beinert (1974) reported the appearance of a pH-dependent g, = 2.6 signal in partially reduced enzyme which, on the basis of a correlated loss of high-spin signal content, was proposed to represent a low-spin equilibrium form of cytochrome a,. The authors noted, however, that the correlation was not quantitative, consistent with the possibility that the signal was, in fact, due to cytochrome a.
Biochemistry, Vol. 30, No. 3, 1991 847
1
A
.76
.66 0
.a
1
.5
B
1.5
Mlnutes
A-
2 cCN; PH 6.7
Resting, pH
8.4
.6
380
440 460 480 Wavelength (nm)
400 420
500
FIGURE 8: Cyanide binding kinetics of resting enzyme 30 s after titration to pH 6.7. An enzyme solution at pH 8.4 and 9.0 M Mheme a was prepared in a buffer containing 50 m M K2HP04.KOH, 167 mM K2S04, and 0.1% (w/v) DM. (A) Low-pH phosphate titrant (see Materials and Methods) was added rapidly, with stirring, to bring the pH to 6.7. After 30 s, partially neutralized sodium cyanide was introduced into the sample with continued stirring to give a final CNconcentration of 25 mM, as described under Materials and Methods. The notation A, denotes the absorbance just prior to cyanide addition. Mixing time (the time between the first and second data points) was approximately 6 s. (B) The spectrum denoted "Resting, pH 8.4" was recorded just prior to the low-pH transition, and the spectrum labeled "+CN-, pH 6.7" was recorded after 15 min of total incubation time with cyanide present.
8.8 was therefore selected for the pH reversal experiment in Figure 7 to ensure that the biphasic kinetics reflected only pH-dependent effects on cytochrome a3 without additive contributions from cytochrome a. Accordingly, the extinction data shown for the pH 10.1-10.5 cases in Figures 4B and 5 reflect observed values that do not take into account the small loss of Soret intensity due to the incremental effect of high pH on cytochrome a. Relationship of Biphasic Kinetics to the Fast and Slow Cyanide Binding Conformers. Earlier observations (Baker et al., 1987) showed that there are two effects of low-pH incubation on resting enzyme: (1) a blue shift in the Soret band; (2) a change in cyanide binding reactivity. Stock enzyme preparations that are purified and maintained at pH 8.6 or higher show a single, rapid phase of reactivity with cyanide, characteristic of the mitochondrial bound state (Baker et al., 1987; see also Materials and Methods). Exposure of detergent-solubilized enzyme to lower pH values will induce the fast reactive form to convert to a more slowly reacting species, thereby resulting in biphasic cyanide binding behavior. Both the actual pH and total time of incubation determine the relative percentages of fast and slow forms present. Although the low-pH-induced shift in the Soret band is clearly biphasic (Figure 7A), data in Figure 8A show that it is not the result of a correspondingconversion to the slow cyanide binding form. The addition of cyanide 30 s after titration to low pH (using conditions similar to those in Figure 7A) resulted in cyanide binding kinetics that were still 100% fast (/cobs = 0.02 s-]).
848 Biochemistry, Vol. 30, No. 3, 1991
Papadopoulos et al.
Additional experiments have established that conversion to the slow, cyanide-reacting form requires longer times of incubation.8 Comparison of Figures 7A and 8A shows that a 2-3-nm low-pH-induced blue shift in the Soret band occurs prior to any fast to slow conversion in cyanide binding behavior. The isosbestic point must therefore arise from two forms of cytochrome u3 that do not differ in their cyanide binding reactivity. This conclusion is also consistent with equilibrium observations. A pH transition from 8.8 to 10.5 will reversibly shift the Soret band 3-4 nm to the red, resulting in a stable band position at 427 nm. Although an isosbestic point accompanies the shift (for example, Figure 2A), the cyanide binding kinetics remain 100% fast at all pH values within this range, even after several hours of incubation (data not shown). The lack of a kinetic correlation in Figures 7A and 8A assumes that cyanide reacts with 100% of the u3centers under our conditions. Figure 8B compares the spectrum of resting enzyme at pH 8.4 (Soret maximum at 422.4 nm) with that of the cyanide-bound form at the same pH. The difference in absorbance at 428 nm between the two forms was determined to be 0.096 from Figure 8A,B. Studies by Van Buuren et al. (1 972) showed that complete reaction of resting enzyme with cyanide led to an absorbance difference of 0.1 16 for the same heme a concentration. Although the pH in their experiments was comparable to ours (pH 8.0 compared with 8.4), the Soret band of their resting enzyme was slightly more blue-shifted, at 419-420 nm. A larger change in absorbance at 428 nm due to cyanide binding is therefore expected. Given this result, it is likely that the cyanide reaction shown in Figure 8A represents nearly 100% of the u3 centers.
DISCUSSION The variability in the position of the Soret band from one preparation to the next is likely to be due to two factors: (1) the actual pH following detergent extraction of the enzyme and (2) the total time of incubation required to obtain purified enzyme as a concentrated stock. Lower pH values and longer incubation times will result in a more blue-shifted Soret band. For example, if no hydroxide is added during the ammonium sulfate fractionation of the cholate extract [following essentially the method of Hartzell and Beinert (1974)], then the pH, in our hands, is observed to settle at 7.5-7.6. The purified enzyme stock is then found to have a Soret maximum at 419 nm, rather than the 423 nm that we regularly observe if the cholate extract is maintained at pH 8.6-9.0 during the fractionation [data not shown; but see Baker et al. (1987)l. Other experimental factors, not yet identified, might also influence the enzyme response to low pH, such as heme u and detergent concentration. However, the common use of ammonium sulfate (an acid), the regular practice of using pH paper to monitor pH, and, in some cases, the inclusion of time-consuming protocols, such as dialysis, probably contribute to the widely diverse Soret band positions that are seen with different purification methods. Variability within the same method is also likely to depend on variations in both incubation time and actual pH. The results in the current investigation are consistent with a proton binding site that can modulate the near-UV absorption properties of cytochrome u3. Figure 7A shows that titration to low pH induces a blue shift in the Soret band that could be reversed by restoring the pH to its initial value. Both the shape and amplitude of the Soret band are recovered. ' S . A. Walter, P. G . Papadopoulos, L. Weng, and unpublished results.
B. M. Baker,
'ImH-a,
(lll)-]+L-
+
H+
I? 4 ImH-a, (III)-]+L-
hm,,-430nm
fast
'DmH-a, (III)-]+LH
I? ii +
H+
ImH-a,(lll)-]+LH
h,,-414nm
Proposed model to explain the pH-dependent shift in the Soret band position. Details are provided in the text. FIGURE 9:
Combining the MCD data in Figure 3 with the absorption spectra shown in Figure 2 documents that the pH-induced shift in the Soret band is associated with an isosbestic point that reflects two different states of cytochrome u3. The absorption due to cytochrome u is stable over the entire pH range except for a minor, reversible loss of Soret amplitude that appears to correlate to the onset of a g, = 2.6 EPR signal [see text for Figure 7; also see Baker and Palmer (1987)l. Following a change in pH, the Soret band eventually stabilizes, showing an apparent equilibrium response to pH that is indicated by the titration profile in Figure 6 . Nonlinear least-squares fitting to the scatter plot, assuming a single acidfbase group, gives an apparent pK, of 7.8. The two states that are associated with the isosbestic point during the approach to equilibrium are proposed to be protonated and deprotonated states of cytochrome u3,having near-UV absorption maxima at approximately 414 and 430 nm, respectively (Figure 4A,B). The summation of their relative intensities at any selected pH results in an observed Soret position between 416 and 427 nm. Another cyanide-sensitive absorption band at approximately 410 nm provides additional spectral evidence for cytochrome u3heterogeneity, but, unlike the bands at 414 and 430 nm, it does not respond to changes in pH. The time course associated with a pH-induced shift in the Soret band was reversibly biphasic (Figure 7A), despite retention of an isosbestic point (Figure 7B,C). The reaction u3 + H+ + u3.H+, which is implied by the isosbestic point, is expected to respond to a pH transition by undergoing a simple, first-order approach to a more stable equilibrium distribution that depends on the final pH. The biphasic reactivity in Figure 7A can be explained if there are two optically identical states of cytochrome u3 that bind protons at different rates. Accordingly, the two electronic states that give rise to the isosbestic point simply reflect whether or not the proton is bound to the u3 site and not on factors that affect proton reactivity. This point will be discussed again in the context of a model that is shown in Figure 9. Support for a proton binding site near cytochrome u3 also comes from the deuterium isotope effect that was observed by Schoonover et al. (1988). Resonance Raman studies showed that exchange of resting enzyme (Soret maximum, 423-424 nm) into D,O buffer at pD -8.0 induced a 4 cm-' shift in a low-frequency band (from 223 to 219 cm-I) that was assigned to the Fe(II1)-N(His) stretch of cytochrome u3. There are several ways that pH can influence the electronic structure of cytochrome u3. One effect expected at alkaline pH is a transition from high to low spin. Cyanide reacts with resting enzyme to produce a red-shifted derivative that has a Soret maximum at 428 nm. A similar red shift to 427 nm
Spectral Effects of pH on Cytochrome Oxidase occurs following a 2-3 h exposure to pH 10.1-10.5 (Figures 1 and 2A). However, the similarity in the band positions of the cyanide and alkaline derivatives cannot be used as an indicator of a common structural change in cytochrome u3, such as conversion to low spin. As discussed previously, the near-UV absorption band shape and intensity of these derivatives are quite different (Figure 1). Interestingly, the alkaline-treated case shows a clear transition at 430 nm, following subtraction of cytochrome u (Figure 4A,B), which is much more red-shifted than expected for a high-spin ferric heme u complex, but is within the range expected for low-spin or reduced forms. Despite this optical indicator, MCD and magnetic susceptibility data in Figure 3 indicate that there is no transition to low spin and no reduction of either cytochrome, as previously reported by Baker and Palmer (1987) and Baker et al. (1987). [The region at 450 nm is very sensitive to the oxidation state of cytochrome uu3 (Babcock et al., 1976).] It should also be mentioned that the 655-nm charge transfer band, thought to be a marker for high-spin cytochrome u3 (Palmer et al., 1976), appears to lose intensity in red-shifted enzyme (data not shown). Visible MCD, however, shows that the transition simply shifts underneath the a-band, making it more difficult to detect in the visible absorption spectrum (Baker et al., 1987). A red shift in the Soret band of ferric heme is normally associated with an increase in electron donation from the axial ligands, resulting from either an increase in ligand basicity or a reduction in steric constraints (Wang & Brinigar, 1979; Martinis et al., 1989). The difficulty with this explanation is that such effects are likely to force a low-spin transition. For example, removal of the N ( 1) proton from the proximal histidine of cytochrome u3 seems unlikely since imidazolate (Im-) in 6-coordinate model heme compounds is often associated with low-spin Fe(II1) (Nappa et al., 1977). Furthermore, George et al. (1 964) have shown that N ( 1) proton of imidazole complexed with ferric metmyoglobin has an ionization constant of 10.3, which is significantly higher than the value of 7.8 found for cytochrome u3 (Figure 6). Perhaps the most compelling argument against changes in axial bond strength resides in the magnitude of the alkaline-dependent red shift (Figure I ) , which is comparable to that induced by cyanide. If the extent of the red shift were simply related to the strength of the axial ligand field, then high-pH conditions, like cyanide, should induce conversion to low spin. Resonance Raman studies have provided insight into the axial bond effects of high pH. Schoonover et al. (1988) have focused on a resonance Raman comparison of two partially protonated states having Soret maxima at 424 and 417 nm, obtained at pH 8.0 and 6.7, respectively. (Reference to Figure 5 indicates that these band positions reflect a change in about 60% of the u3 centers.) Low-pH incubation caused a shift in a low-frequency mode, from 223 to 220 cm-’, that was assigned to the Fe(III)-N(His) stretch of cytochrome u3. Although, definitive data are lacking, several studies suggest that a 3 cm-’ Raman shift reflects only a minor change in the axial bond strength of the proximal histidine. For example, a pH-dependent 3-6 cm-’ shift in the Fe(I1)-N(His) stretch in a variety of plant tissue peroxidases was found by Teraoka et al. (1983) to have no associated effect on the Soret absorption band. Schoonover et al. (1988) found no other shifts in the 200-450 cm-] region that would implicate an effect of alkaline conditions on the bridging ligand. Furthermore, resonance Raman and EPR evidence indicates that there is no decoupling of cytochrome u3 and Cu, at high pH and therefore no change
Biochemistry, Vol. 30, No. 3, 1991 849 in coordination number, unless samples are subjected to prolonged incubation at pH values above 10.5 (Baker & Palmer, 1987), in which case deviations from the isosbestic point are observed (Figure 2). Pending more detailed resonance Raman comparisons, the available data do not point to either the proximal histidine or the bridging ligand as the site of perturbation in cytochrome oxidase. The data are also not consistent with a pH-dependent effect on the formyl substituent of cytochrome u3 since the 1676 cm-I marker for the u3formyl stretch remains unchanged over the pH range 6.7-8.0, despite a 6-7-nm shift in the Soret band position (Schoonover at al., 1988). The combined optical, MCD, and R R data are not consistent with pH-dependent changes in axial bond strength as the origin of the Soret band shift. As an alternative, we propose a model in which a noncoordinatingamino acid residue is able to induce or suppress a point charge in the vicinity of the heme as the pH is raised. Davis et al. (1981) showed that protonating a nonconjugating amine group on the periphery of a chlorophyll macrocycle could induce a 4-nm blue shift in the red absorption maximum. A negative charge was presumed to have the opposite effect. Although no effect on the Soret band position was observed, the possibility remains that charges at different positions around the porphyrin could have strikingly different effects. Similarly, Lanyi et al. (1988) have shown that anion binding to site I1 in halorhodopsin induces a red shift in the absorption maximum. A model based on this “through space” interaction is shown in Figure 9. At high pH, an amino acid residue labeled “L” is deprotonated and is presumed to have a negative charge that can electrostatically couple with the negative positive charge on the Fe(1II)-porphyrin complex. (The “L” site might be a tyrosine residue, as discussed below.) The coupling is proposed to stabilize the excited state (T*)for the optical transition dipole, leading to an absorption band at 430 nm. At low pH, the “L” site is protonated and loses its negative charge. Without the electrostatic interaction, the excited state is destabilized, and a new transition at 414 nm is observed. The difference between the 414- and 430-nm states corresponds to an interaction energy of about 2.5 kcal/mol of heme a. (An equivalent model would depict an “L” site that had a positive charge at low pH which became neutral at high pH, such as a histidine.) Weak coupling of the Soret resonance to the Fe(II1)-N(His) stretching mode would account for the small Raman shift that Schoonover et al. (1988) observed. Subunit topography studies of Holm et al. (1987) suggest that the protonatable residue may be a tyrosine. Transmembrane segment VI of subunit I has an invariant histidine (His-273) that was proposed to form the proximal ligand of cytochrome u3. A conserved tyrosine residue, also present in segment VI, might be close enough to the a3 site to promote the high-pH-dependent electrostatic coupling shown in Figure 9. Stabilization of the negative phenoxide through coupling would also provide a mechanism to lower the pK, of tyrosine from its usual value of 10.1. Theoretical calculations, based on the structural model of Holm et al. (1 987), that examined the effect of external point charges on the Soret band would be of interest. The model shown in Figure 9 must also accommodate the fast and slow reactive phases in Figure 7A. As a working hypothesis, the biphasic kinetics are presumed to reflect two different protein conformations that allow different proton access rates to the u3 site. The conformations, denoted “fast” and “slow” in Figure 9 (top and lower halves, respectively), must have identical near-UV absorption spectra in order to
850 Biochemistry, Vol. 30, No. 3, 1991 explain the retention of an isosbestic point following a pH transition (Figure 7B,C). We hypothesize that only the protonated and deprotonated forms of site “L” are optically different and that these forms give rise to the isosbestic point. Interestingly, Wilson et al. (1980) have found the Soret absorption to be very similar for bovine and elasmobranch cytochrome oxidase, despite differences in aggregation state and subunit composition. Dimer and monomer might influence differently the rate at which solvent protons diffuse to the a3 site without themselves being spectrally different. Baker et al. (1987) observed that high enzyme concentrations (600 pM heme a ) prevented the low-pH-induced blue shift in the Soret band, possibly due to protein aggregation. Work is now in progress to determine if other conditions that have been reported to change aggregation state, such as ionic strength and detergent concentration, will correspondingly change the amplitudes of the fast and slow phases shown in Figure 7A. Proton interactions in the resting form of cytochrome oxidase may be related to the physiological properties of the enzyme. Although the fast phase in Figure 7A is still much slower than enzyme turnover times, Jones et al. (1984) found that cyanide binding to oxidized cytochrome a3 was more than IO5 times faster in partially reduced enzyme than it was in resting or pulsed enzyme. It is possible that a similar effect would be observed for proton binding, but no evidence for this is currently available. Nonetheless, the results presented here provide new information on the pH-dependent behavior of cytochrome a3 and provide evidence for a proton binding site, possibly a tyrosine residue, that can change the position of the Soret band by as much as 11 nm. The possibility that the 414and 430-nm forms of cytochrome a3 constitute a “switch” that could assist proton pumping during enzymatic turnover is an intriguing possibility warranting further study. ACKNOWLEDGMENTS We gratefully acknowledge helpful discussions with Professors s. Ferguson-Miller, w. A. Cramer, G. Palmer, and Dr. J. Schoonover. We also thank Professor Roy Mason in the Chemistry Department at Northern Illinois University for the use of his MCD spectrometer. Registry No. Cytochrome oxidase, 9001-16-5; cytochrome a3, 72841-18-0; cyanide, 57-12-5.
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