ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978
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Indirect Coulometric Titrations of Complex IV in Intact Mitochondria and Submitoc hondriaI Particles Robert Srentirmay' and Theodore Kuwana" Department of Chemistry, The Ohio State University, Columbus, Ohio 432 10
The complex I V region of intact mitochondria or submitochondrial particles was studied using electrogenerated viologen radical cation for reductant and molecular O2 as oxidant by the indirect coulometric titration method (ICT). Doublewavelength modulation specrophotometry was employed to circumvent the problems of solution turbidity and low redox component concentrations. The redox potentials for components evaluated using ICT were in good agreement with previous reports. I t was also demonstrated that redox information could be obtained using ICT generation of reductant or oxidant in the presence of mediators for acquisition of potentiometric data. Best fit potential data from Minnaert plots gave values of redox potentials for cytochrome c oxidase which were in good agreement with those obtained from purified materials.
At our present level of understanding of the respiratory chain, it is well established t h a t any study directed toward elucidating energy transduction processes must be carried out utilizing intact membrane particles ( 1 ) . The more intact the sample is, the more efficient the energy utilization process. In order to prepare submitochondrial fractions with the capabilities of A T P generation, special preparation procedures must be followed (2). T h e critical role of membrane topography and vectorially directed chemical reactions has again been re-emphasized in a series of recent reviews concerning oxidative phosphorylation and photophosporylation ( I ) . In addition, cytochrome oxidase manifests in the intact system a number of properties which are not observed in the isolated preparations. T h e highest oxygen turnover rates have been reported in the intact system ( 3 ) . In the intact membrane, evidence has suggested t h a t the enzyme exists in an asymmetric transmembrane orientation. Heme a3 has been identified reacting with oxygen on the matrix side of the inner membrane, while heme u is found with cytochrome c in the inner membrane space ( 4 ) . Rapid oxidation of oxidase and the c cytochromes is accomplished in the intact mitochondria by synchronous proton releases (1). The apparent mid-point potential for heme a j or heme a , or both has a significant dependence on the phosphate potential and p H only in t h e intact system ( 3 ) . In addition, effects of A T P on both the spectroscopic and ligation properties of heme u3 have been reported ( 3 ) . Unfortunately in working with the intact systems, a number of experimental difficulties arise. Because of the high turbidity of the samples and the low relative cytochrome concentrations, the concentrations of cytochromes c and oxidase in mitochondrial and submitochondrial preparations for visible spectroscopic studies are typically only 1to 3 WM. This results in a considerable sacrifice of experimental precision for these studies. In addition, one must contend with the possible 'Present address, Shell Development co., P.O. Box 1380, Houston, Texas 77001. 0003-2700/78/0350-1879$01.00/0
interference and complications arising from the additional membrane components and exogenous species present in the preparations. Variations also occur in the activities and properties of different preparations. Most importantly for the purposes of redox studies, it is often difficult to poise the respiratory enzymes at well defined redox states because of continuous reduction of the respiratory chain by substrates naturally found in the preparations. It is also difficult to assess whether true equilibrium is ever achieved in such complex multiphase systems. This portion of our research was undertaken to investigate whether the Indirect Coulometric Titration, ICT, method could be utilized to study intact membrane systems. No such investigation had been made previously. However, a number of reports have shown the feasibility of coupling intact membrane systems t o electrochemical methods utilizing mediators (6-11). In particular, t h e extensive work of D. L. Wilson and P. L. Dutton (12, 13) has shown the reality of obtaining valuable potentiometric information on the respiratory components utilizing a judiciously chosen set of redox mediators to couple t h e platinum-sensing electrode to the enzymes. I n this way, a well recognized and important thermodynamic picture of the respiratory components has evolved (12, 13). Our first objective in this research was to test the ICT method to find out whether quantitative redox information could be obtained on the complex IV region of the chain. Then we wanted to compare this information to data obtained by different methods. Our second objective was to test whether the ICT method could be coupled to the potentiometric technique. The experimental versatility, ease of application, and accuracy of the technique, as previously found in our laboratory with purified enzymes, suggested that the ICT method might be a n extremely powerful technique for biochemists in studying the redox and kinetic properties of intact biological systems as well.
EXPERIMENTAL Intact washed Nagarse beef heart mitochondria (HBHM) were a gift of G. P. Brierly of the Department of Physiology of The Ohio State University. HBHM suspensions, 44 mg/mL and 25 mg/mL, were stored in 0.25 M sucrose, 0.01 M Tris at pH 7.4, frozen at -10 "C and were thawed only immediately prior to use. Mediator and enzyme solutions were prepared fresh just prior to use for each experiment. Electron Transport Particles (ETP) were prepared from HBHM according to the following procedures. HBHM (25 mg/mL) was sonicated in two 30-s bursts in 30-mL batches at high power. The samples were then centrifuged for 30 min at 12 000 rpm using an SS 34 Sorvall. The supernatant was then decanted and centrifuged at 40000 rpm for 30 min. Pellets from the second centrifugation were resuspended in Tris-pH 8 buffer to original volume and homogenized using a Teflon homogenizer. ETP samples prepared in this fashion were found to be relatively unstable for quantitative redox titrations. The cytochromes were completely autoreduced by residual substrates in approximately 90 min. I3TP samples, prepared according to the initial procedure, were then resonicated for two 30-s bursts, again centrifuged at 40000 rpm for 30 min, and then the pellets redissolved to original volume. This procedure was repeated once more so that all samples were sonicated at least C 1978 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978
three times and resuspended three times. E T P particles redissolved in this fashion were found to be stable for redox titration for periods of 4 to 6 h after initial thawing before the onset of slow autoreduction. All preparative procedures were performed in ice baths at 0 "C. Samples were stored frozen at -10 "C. Once autoreduction set in, the stability of the samples could not be recovered by freezing and reuse. One cell for potentiometric work was constructed similarly to that described previously (141, except that a 1.25-cm diameter cavity was bored out as the working cell cavity and a platinum flag (1 cm X 1 cm) was incorporated for potentiometric measurements. A second cell of larger volume (9.45 mL) was constructed of similar design for quantitative titrations. All valves and windows were epoxied into position. Sample and mediator preparations and procedures including solution deoxygenation were identical to those described previously (14, 15). All experiments were performed in phosphate buffer pH 7.0 and w = 0.15 at 50-mM sucrose levels. Spectroscopic and electronic equipment used were the same as described previously (24-16). The sample cell was moved near the P M T housing approximately 1 cm from the PMT front surface. For all experiments, no reference sample was used. Baseline corrections to spectra were made utilizing the NOVA 800 minicomputer on a real-time basis. After an initial spectrum of the fully oxidized respiratory chain was acquired into computer memory, all subsequent spectra were plotted as baseline corrected by the computer utilizing the Fortran program TITREPI (17,18). Thus, difference spectra were plotted out directly during the course of the experiment. All raw data, however, was stored on disc memory for future analysis and comparison.
RESULTS AND DISCUSSION Attempts t o poise and quantitatively reduce and oxidize t h e cytochromes of intact mitochondria without potentiometric redox buffers proved unsuccessful. Utilizing electrogenerated methyl viologen radical cation, MV'., and O2 as mediator titrants (291, the I C T method could readily be applied for reducing and oxidizing the cytochromes to appropriate redox levels including complete oxidation. The order of reduction and oxidation was exactly that expected on the basis of t h e thermodynamic E"' values of the components. T h a t is, optical components were reduced in the order cyto a3 a, c, c1 bT, bK excess MV'.. Reduction of the entire respiratory chain by endogenous substrates occurred in 10 to 30 min, depending on how long the sample was maintained under anaerobic conditions. T h e longer the experimental sample was analyzed, the faster autoreduction occurred, even in the presence of 8 wg/mg protein of Antimycin or Rotenone. Stability to autoreduction was considerably improved in the presence of potentiometric redox buffers. Spectra and correlations, as shown previously (16),could be obtained for approximately the first 2 or 3 h after rendering the solution anaerobic. T h e autoreduction rates for the samples depended on the potential and at potentials approximating the E"' (f60 mV) of the potentiometric mediators, rates were approximately -1 mV per 1 t o 10 min for 50 to 100 pM mediator concentrations. In addition, a slow hysteresis was consistently observed for titrations where the b cytochromes were being titrated. Equilibrium appeared to require 10 to 30 min before t h e redox levels of the c and b cytochromes reached a steady state. T h e potentivmetric data obtained in the experiment discussed above are shown in Figure 1. These data essentially agree with the results of Dutton, Wilson, and Lee (6) for the complex IV region, except that the a and c cytochromes appear to be 15 to 30 mV higher in potential. T h e curvature in the potentiometric data a t lower potentials (150-250 mV) may be the result of these hysteresis effects or poor mediation to t h e b cytochromes which are imbedded within the mitochondrial membrane ( 4 ) . A computer simulation drawn through the cytochrome oxidase data represents the best fit (*lo mV) as determined by visual inspection of simulation
Figure 1. The oxidation ( 0 ,A) and reduction ( 0 ,A) potential dependence of the 605-620 nm ( 0 ,0 ) and 551-540 nm ( A , A) absorbance changes of intact mitochondria suspended at 2.2 m g / m L protein at pH 7.0 in phosphate buffer with 50 m M sucrose. Potentiometric mediators added were hydroquinone (80 pM), duroquinone (50 pM), Fe(CN);(100 pM). PMS (40 pM), CoQ, (50 pM). MV = 1.2 mM. Solid lines are for computer simulations for (a) cyto c, n = 1.0, Eo' = 255 mV; (b) heme a", n = 1.0, E"' = 350 mV (50/50 model); (c) low potential heme, n = 1.0, E"' = 215 mV; (d) low heme, n = 1.0, Eo' = 240 m V . Oxidation was with electrogenerated oxygen
- - -
v Flgure 2. Sequential computer calculated difference spectra obtained for submitochondrial particles (1 .O mg/mL) in phosphate buffer at pH 7.0 in 50 m M sucrose. Spectra were taken during the course of an ICT reduction with BV' (1.0 mM). (A) reference A = 540 nm; (B) reference A = 562 nm; (C) reference A = 620 nm
curves. Both curves d and c merge in the high potential region and fit the simulated curve labeled as curve b in this region. The 50/50 percent absorbance model for the two hemes was assumed. Again, this agrees with the observations of D. F. Wilson (6, 12, 23). Submitochondrial particles sonicated and centrifuged only once at a high rate (40000) showed an approximately threefold improvement in redox stability. When samples were carried through the sonication and washing procedure three times, a considerably high degree of stability was achieved. Redox states for the complex IV region of the chain could be set and poised for as long as 15 min with no noticeable spectral change (&0.001 au). In addition, if excess O2 was generated in the cell, it had to be reduced quantitatively by MV'. or benzylviologen radical, BV'. before reduction of oxidase could be initiated. A sample of the type of difference spectra that could be obtained during a reductive titration is shown in
ANALYTICAL CHEMISTRY, VOL. 50,
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Figure 3. Single wavelength monitor of the cytochrome oxidase peak during the course of an ICT reduction with MV'. (1.0 mM) and subsequent oxidation with electrogenerated 0,. Derivative spectroscopy (dau = derivative absorbance unit) with a 10-nm peak to peak sine wave modulation was used (76). Submitochondrial particles were at 1.O mg/mL protein concentration
Figure 4. AA-q plots for quantitative ICT reduction of submitochondrial particles (1 .O mg/mL) with MV'. (1.5 mM). (0)605-620 nm, 100% AA = 0.033 au; (A) 551-540 nm, 100% AA = 0.032 au; (0) 562-575 nm, 100% AA = 0.028 au. A 9.44-mL cell with 1.25-cm path length was used
Figure 2, where part A shows the original spectra obtained utilizing 540 nm as the reference wavelength. These spectra were used to quantitate the c c1 components (552-540 nm). T h e computer was utilized to replot the data, as shown in Figure 2, parts B and C, with respect t o the wavelengths of 575 nm and 620 nm, respectively. These data plots allowed for the quantitiation of the b (562 nm) and oxidase (605 nm) components with respect to their individual isosbestics. Figure 3 shows the absorbance changes observed for the cytochrome oxidase cy peak during the course of a titration. T h e derivative spectroscopic approach was used for this monitoring, since the high frequency modulation (500 Hz) allowed for continuous monitoring while stirring. In this case, a calibration run indicated that a change of 0.0010 a u in the derivative spectrum (10 nm peak-to-peak sine wave modulation) a t the N band corresponded to 0.0014 au in t h e dual wavelength mode. In these cases, titration through the b cytochrome region resulted again in a redox hysteresis similar to that described above. In Figures 4 and 5 are plotted the absorbance vs. charge data for cytochromes aa3,c + cl, and bT + bK obtained during the course of reduction with MV'. and oxidation with 0%The charge axis was normalized t o equivalents per mole oxidase, where the oxidase concentration was calculated using a of 24 m M for the enzyme a t 605-620 nm. Ideally, for complex IV, t h e extrapolated end point should have required approximately 5 equivalents, since the oxidase (-1.1 pM) and c cytochromes ( - 1.2 b M ) were found approximately
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13, NOVEMBER 1978
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