Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6497-6501, July 1992
Biophysics
Photoinduced electron transfer from tris(2,2'-bipyridyl)ruthenium to cytochrome c oxidase (photoreduction/copper ion CUA/cytochrome a)
THOMAS NILSSON Department of Biochemistry and Biophysics, Chalmers University of Technology and Goteborg University, S412 % Giteborg, Sweden
Communicated by Harry B. Gray, April 1, 1992
ABSTRACT Flash photolysis has been used to effect electron transfer from tris(2,2'-bipyridyl)ruthenium(I) to cytochrome c oxidase in the presence of a sacrificial electron donor, aniline. The observation that photoreduction occurs only at low ionic strength and high pH indicates that an electrostatic complex between the ruthenium compound and the enzyme is the reactive species. The reaction was followed at 830, 605, and 445 nm. The initial absorbance changes observed suggest that the copper ion CUA is the preferred electron acceptor and that electron transfer from the excited ruthenium complex takes place in 90% as determined from the absorbance at the peak at 607 nm in the difference spectrum and a Ae(607-630 nm) of 11 mM-1 cm-1
(15).
Preparation of the Ferryl Intermediate (Compound F). Compound F was prepared by reaction of the oxidized enzyme with excess hydrogen peroxide as described (16). Oxidized enzyme (12 ,uM) was incubated with hydrogen peroxide (5 mM) and the reaction was followed by difference spectroscopy. After 30 min, the characteristic spectrum of compound F (15) with a broad peak at 582 nm and a minor peak at about 530 nm had developed. There was no peak present at 607 nm and the spectrum was stable for at least another 20 min. The yield of compound F was 70-80%6 as determined from the absorbance difference at 582 nm and a Ae(582-630 nm) of 5 mM-1-cm-1 (15). Flash Photolysis Monitored at 605 or 445 nm. Monitoring light obtained from a 250-W tungsten/halogen lamp was passed through a heat filter and an interference filter (445 or 605 nm) or a monochromator and then focused on the sample cuvette (10 x 10 mm). The transmitted light was focused on the entrance slit of a double monochromator and detected at the exit slit by a photomultiplier (Hamamatsu R269; Middlesex, NJ) operated as described by Porter and West (17). The output was amplified (Hamamatsu C1053 preamplifier followed by a Tektronix AM502 differential amplifier; offset voltage was provided by a Tektronix PS505-1 voltage supply) and recorded with a transient digitizer (Biomation 2805; Santa Clara, CA) interfaced to a microcomputer. A frequency-doubled neodymium-yttrium/aluminum garnet (NdYAG) laser (Quantel, Santa Clara, CA) was used for photolysis. The wavelength was 532 nm, the duration of the pulse was 9 ns, and the total energy was -200 mJ (unless otherwise stated). The photolysis light was directed at 900 to the monitoring beam. Flash Photolysis Monitored at 830 rn. Monitoring light at 830 nm was obtained from a diode laser and detected using a photodiode as described by Hoganson et al. (18), except that the detector was covered with an 830-nm interference filter. Photolysis conditions were the same as above. All photolysis experiments were carried out at room temperature (22°C).
RESULTS Fig. 1A shows the absorbance changes at 605 nm induced by flash photolysis of tris(2,2'-bipyridyl)ruthenium and cytochrome oxidase in the presence of aniline at low ionic strength. Photolysis results in an absorbance increase taking place in two kinetically distinct steps: an almost instantaneous increase followed by a slower phase. The initial phase is not resolved because of scattered light from the laser flash and the strong luminescence of the ruthenium complex at 605
uL
0
(0
0
0.1 0.05 Time, ms
0.15
0.2
FIG. 1. Absorbance changes at 605 nm induced by flash photolysis of cytochrome oxidase in the presence of tris(2,2'-bipyridyl)ruthenium and aniline. Trace A was recorded from a sample of 18 ,uM enzyme, 80 ,uM tris(2,2'-bipyridyl)ruthenium dichloride, and 10 mM aniline in 5 mM Tris/acetic acid buffer (pH 8.1) containing 0.1% dodecyl maltoside. Trace B was obtained from a sample of the same composition except that 0.5 M NaCl was added. Both traces are averages of 20 transients.
nm (life time, 0.6 ,us; ref. 5). For the second phase, a rate constant of 2.1 x 104 s-1 was obtained. The contribution of the slower phase to the total amplitude was found to be "85%. At increased ionic strength (Fig. 1B), no flashinduced absorbance changes were observed. Lowering the pH to 7 also abolished the signal (data not shown). Similar time courses were observed at 445 nm (Fig. 2). The time resolution is not sufficient for determination of the rate constant of the rapid phase in the trace obtained at low ionic strength. For the slower phase, the rate constant obtained was 2.3 x 104 s-1 and its amplitude was 480% of the total
amplitude. The data shown in Figs. 1 and 2 were collected using different enzyme concentrations to obtain good signal-tonoise ratios at both wavelengths. Since the resulting difference in the optical density at the wavelength of the laser flash may cause different yields of the excited ruthenium complex,
AA =0.005 A
0
0.05
Time,
0.1 ms
0.15
0.2
FIG. 2. Absorbance changes at 445 nm induced by flash photolysis of cytochrome oxidase in the presence of tris(2,2'-bipyridyl)ruthenium and aniline. Both traces were recorded under the conditions given in Fig. 1 except that the concentration of cytochrome oxidase was 8 izM.
Proc. Natl. Acad. Sci. USA 89 (1992)
Biophysics: Nilsson data at 605 nm were also collected from a sample composed as in Fig. 2 for comparison of the absorbance changes at 605 and 445 nm. From these results, a value for AA605/A44A (total amplitudes) of -0.3 was obtained. The value expected for reduction of cytochrome a only is 0.27-0.36, whereas the corresponding figure for cytochrome a3 is 0.043-0.073 (19, 20). It thus seems likely that the major part ofthe signal is due to cytochrome a reduction. The wavelength dependence of the absorbance change in the Soret region is shown in the kinetic difference spectrum in Fig. 3. Comparison with the static difference spectra of cytochromes a and a3 (20) supports the conclusion that cytochrome a is reduced. By using a AE605(red-ox) of 16 mM-1'cm-1 for cytochrome a (19), it can be calculated that the yield of reduced cytochrome a after a flash corresponds to -3% of the total protein. The observation that the yield was roughly proportional to the flash energy and did not increase upon a 10-fold increase in the concentrations of cytochrome oxidase and ruthenium complex (using a 1-mm cell) suggests that it is limited by the yield of the excited ruthenium complex rather than incomplete binding of the complex to the protein. The finding that the rate constant for the second phase is similar to values reported earlier for the equilibration between CUA and cytochrome a (10, 11) suggests that it could be due to electron transfer from CUA. To investigate this possibility, data were recorded also at 830 nm where the oxidized form of CUA absorbs. Fig. 4 shows a transient decrease in absorbance, corresponding to CUA reduction, followed by a slower phase of reoxidation. The initial absorbance decrease takes place in
Biophysics
Photoinduced electron transfer from tris(2,2'-bipyridyl)ruthenium to cytochrome c oxidase (photoreduction/copper ion CUA/cytochrome a)
THOMAS NILSSON Department of Biochemistry and Biophysics, Chalmers University of Technology and Goteborg University, S412 % Giteborg, Sweden
Communicated by Harry B. Gray, April 1, 1992
ABSTRACT Flash photolysis has been used to effect electron transfer from tris(2,2'-bipyridyl)ruthenium(I) to cytochrome c oxidase in the presence of a sacrificial electron donor, aniline. The observation that photoreduction occurs only at low ionic strength and high pH indicates that an electrostatic complex between the ruthenium compound and the enzyme is the reactive species. The reaction was followed at 830, 605, and 445 nm. The initial absorbance changes observed suggest that the copper ion CUA is the preferred electron acceptor and that electron transfer from the excited ruthenium complex takes place in 90% as determined from the absorbance at the peak at 607 nm in the difference spectrum and a Ae(607-630 nm) of 11 mM-1 cm-1
(15).
Preparation of the Ferryl Intermediate (Compound F). Compound F was prepared by reaction of the oxidized enzyme with excess hydrogen peroxide as described (16). Oxidized enzyme (12 ,uM) was incubated with hydrogen peroxide (5 mM) and the reaction was followed by difference spectroscopy. After 30 min, the characteristic spectrum of compound F (15) with a broad peak at 582 nm and a minor peak at about 530 nm had developed. There was no peak present at 607 nm and the spectrum was stable for at least another 20 min. The yield of compound F was 70-80%6 as determined from the absorbance difference at 582 nm and a Ae(582-630 nm) of 5 mM-1-cm-1 (15). Flash Photolysis Monitored at 605 or 445 nm. Monitoring light obtained from a 250-W tungsten/halogen lamp was passed through a heat filter and an interference filter (445 or 605 nm) or a monochromator and then focused on the sample cuvette (10 x 10 mm). The transmitted light was focused on the entrance slit of a double monochromator and detected at the exit slit by a photomultiplier (Hamamatsu R269; Middlesex, NJ) operated as described by Porter and West (17). The output was amplified (Hamamatsu C1053 preamplifier followed by a Tektronix AM502 differential amplifier; offset voltage was provided by a Tektronix PS505-1 voltage supply) and recorded with a transient digitizer (Biomation 2805; Santa Clara, CA) interfaced to a microcomputer. A frequency-doubled neodymium-yttrium/aluminum garnet (NdYAG) laser (Quantel, Santa Clara, CA) was used for photolysis. The wavelength was 532 nm, the duration of the pulse was 9 ns, and the total energy was -200 mJ (unless otherwise stated). The photolysis light was directed at 900 to the monitoring beam. Flash Photolysis Monitored at 830 rn. Monitoring light at 830 nm was obtained from a diode laser and detected using a photodiode as described by Hoganson et al. (18), except that the detector was covered with an 830-nm interference filter. Photolysis conditions were the same as above. All photolysis experiments were carried out at room temperature (22°C).
RESULTS Fig. 1A shows the absorbance changes at 605 nm induced by flash photolysis of tris(2,2'-bipyridyl)ruthenium and cytochrome oxidase in the presence of aniline at low ionic strength. Photolysis results in an absorbance increase taking place in two kinetically distinct steps: an almost instantaneous increase followed by a slower phase. The initial phase is not resolved because of scattered light from the laser flash and the strong luminescence of the ruthenium complex at 605
uL
0
(0
0
0.1 0.05 Time, ms
0.15
0.2
FIG. 1. Absorbance changes at 605 nm induced by flash photolysis of cytochrome oxidase in the presence of tris(2,2'-bipyridyl)ruthenium and aniline. Trace A was recorded from a sample of 18 ,uM enzyme, 80 ,uM tris(2,2'-bipyridyl)ruthenium dichloride, and 10 mM aniline in 5 mM Tris/acetic acid buffer (pH 8.1) containing 0.1% dodecyl maltoside. Trace B was obtained from a sample of the same composition except that 0.5 M NaCl was added. Both traces are averages of 20 transients.
nm (life time, 0.6 ,us; ref. 5). For the second phase, a rate constant of 2.1 x 104 s-1 was obtained. The contribution of the slower phase to the total amplitude was found to be "85%. At increased ionic strength (Fig. 1B), no flashinduced absorbance changes were observed. Lowering the pH to 7 also abolished the signal (data not shown). Similar time courses were observed at 445 nm (Fig. 2). The time resolution is not sufficient for determination of the rate constant of the rapid phase in the trace obtained at low ionic strength. For the slower phase, the rate constant obtained was 2.3 x 104 s-1 and its amplitude was 480% of the total
amplitude. The data shown in Figs. 1 and 2 were collected using different enzyme concentrations to obtain good signal-tonoise ratios at both wavelengths. Since the resulting difference in the optical density at the wavelength of the laser flash may cause different yields of the excited ruthenium complex,
AA =0.005 A
0
0.05
Time,
0.1 ms
0.15
0.2
FIG. 2. Absorbance changes at 445 nm induced by flash photolysis of cytochrome oxidase in the presence of tris(2,2'-bipyridyl)ruthenium and aniline. Both traces were recorded under the conditions given in Fig. 1 except that the concentration of cytochrome oxidase was 8 izM.
Proc. Natl. Acad. Sci. USA 89 (1992)
Biophysics: Nilsson data at 605 nm were also collected from a sample composed as in Fig. 2 for comparison of the absorbance changes at 605 and 445 nm. From these results, a value for AA605/A44A (total amplitudes) of -0.3 was obtained. The value expected for reduction of cytochrome a only is 0.27-0.36, whereas the corresponding figure for cytochrome a3 is 0.043-0.073 (19, 20). It thus seems likely that the major part ofthe signal is due to cytochrome a reduction. The wavelength dependence of the absorbance change in the Soret region is shown in the kinetic difference spectrum in Fig. 3. Comparison with the static difference spectra of cytochromes a and a3 (20) supports the conclusion that cytochrome a is reduced. By using a AE605(red-ox) of 16 mM-1'cm-1 for cytochrome a (19), it can be calculated that the yield of reduced cytochrome a after a flash corresponds to -3% of the total protein. The observation that the yield was roughly proportional to the flash energy and did not increase upon a 10-fold increase in the concentrations of cytochrome oxidase and ruthenium complex (using a 1-mm cell) suggests that it is limited by the yield of the excited ruthenium complex rather than incomplete binding of the complex to the protein. The finding that the rate constant for the second phase is similar to values reported earlier for the equilibration between CUA and cytochrome a (10, 11) suggests that it could be due to electron transfer from CUA. To investigate this possibility, data were recorded also at 830 nm where the oxidized form of CUA absorbs. Fig. 4 shows a transient decrease in absorbance, corresponding to CUA reduction, followed by a slower phase of reoxidation. The initial absorbance decrease takes place in
