BIOCHEMICAL
Vol. 184, No. 2, 1992 April 30, 1992
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1035-1041
EVIDENCE FOR A BAND III ANALOGUE IN THE NEAR-INFRARED ABSORPTION SPECTRA OF CYTOCHROME E OXIDASE ‘Olaf Einarsdktir, Katy E. Georgiadis,and Timothy D. Dawes Departmentof Chemistry, University of California, SantaCruz SantaCruz, California 95064 Received
March
17,
1992
Summary. Ground state near-infrared absorption spectraof fully reduced unliganded and fully reducedCO @+ CUA+g2+-CO CUB+)cymchrome Eoxidasewere investigated. Flash-photolysis time-resolved absorptiondifference spectraof the mixed-valence h3+ CUA~+a32+-C0 CUB+)and the fully reduced CO complexes were also studied. A band near 785 nm (E -50 M-km-l) was observed in the fully reducedunliganded enzyme and the CO photoproducts. The time-resolved 785 nm banddisappearedon the sametimescale(tin -7 ms) asCO recombinedwith cytochrome a32+. This band, which is attributed to the unligandedfive coordinate ferrous cytochrome a2+, has somecharacteristicsof band III in deoxy-hemoglobin and deoxy-myoglobin. A secondband was observed at -710 nm (E -80 M-km-l) in the fully reducedunligandedand the fully reduced CO complexes. This band, which we assignto the low spin ferrous cytochrome 3, appearsto be affected by the ligation stateat the cytochromea32+site. 0 1992 AcademicPress, 1°C.
Optical absorptionstudieson cytochromeoxidasein the near-infraredregion have primarily concentrated on the fully oxidized enzyme, which showsa broad band at 830 nm. This band, which is also present in the mixed-valence CO complex (a3+ CUA~+aj2+-CO CUB+), has been attributed to CUA~+(l-3). The fully reducedunligandedand the fully reduced CO-bound enzyme have no reported absorbancesin this region. However, five coordinate high spin deoxyhemoglobin and deoxy-myoglobin have been shownto have a charge-transfertransition near 760 nm with E -200 M-km-l
(4,5). This transition, designatedband III, has also been observed
following photodissociationof CO from theseproteins (6-8). This bandis absentin the spectraof the six coordinate CO-bound proteins, which show no absorbancesin this region. Based on circular dichroism, magnetic circular dichroism, and polarized single-crystal absorption,Eaton & al. (4) have assignedthis transition to a charge-transfertransition betweenthe porphyrin x system and the iron K system(azu + dzy). Until recently, similar absorbanceshad not beenreported for cytochrome E oxidase (9). In this paper, we report the presenceof a bandat -785 nm in the near infrared spectrumof fully reducedunligandedcytcchrome Eoxidaseand in the time-resolveddifference spectraobtained following photodissociationof CO from the mixed-valence and the fully reducedcomplexes. This band is attributed to the unliganded five coordinate ferrous cytochrome a32+. A secondband is 0006-291X/91$1.50 1035
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observed near -7 10 nm in the ground state infrared spectra of the fully reduced unliganded and the fully reduced CO-bound enzyme. We assign this band to the ferrous low-spin cytochrome a.
mrials and Metho& Cytochmme oxidase was isolated according to the procedure of Yoshikawa ti d. (10). prior to reduction, the oxidized enzyme in 0.1 M Na-phosphate pH 7.4 was filtered through a 0.2 pm filter into a fluorescence sample cuvette (10x4 mm) equipped with a stopcock for enzyme deoxygenation. The deoxygenated enzyme was reduced under N2 with a small excess of sodium dithionite. The fully reduced CO complex was formed by blowing CO over the enzyme solution for 15-30 minutes. The two electron reduced mixed-valence Co complex was obtained by incubating the deoxygenated oxidized enzyme under 1 atm CO for several hours. The CO complexes were characterized by their visible spectra which were recorded before and after the photolysis measurements to ensure that the samples did not change during the measurements. The ground state near-infrared spectra were recorded on an IBM 9420 UV-Vis spectrophotometer. Cytochrome oxidase concentration was determined using an extinction coefficient of the oxidized enzyme at 598, 8.5 mM-lcm-l in total heme, or an absorbance coefficient of 2.3 mM-Icm-1 (reduced minus oxidized) at 830 nm for CuA2+ (1). The extinction coefficients of the near-infrared bands were determined from the baseline-corrected spectra, which were obtained by a subtraction of a multiple point polynomial fit from the sloping absorbance background. The CO-bound complexes were photolyzed using a DCR-11 Nd:YAG laser (532 nm, 80 rnI/pulse, 7ns duration) with a repetition rate of 2 Hz. The photolysis laser beam was partially focused with a cylindrical lens along the 4 mm path and 90” to the probe beam. The probe beam, a high-power Xenon flashlamp of pulse width 2 lrs (1 l), was focused on the sample cell along the 10 mm path and then recollimated and focused on the entrance slit of a spectrograph/ monochromator. The time-resolved near-infrared spectra of the CO-photodissociated enzyme derivatives were recorded at many wavelengths simultaneously using an optical spectrometric multichannel analyzer (OSMA, Princeton Instruments, Inc.) detector. The spectra at various times following photolysis were obtained by varying the time of an intensifier gate pulse (gate width 5 ns to 2.5 ps) relative to the excitation pulse using a delay generator. The spectra were averaged, transferred to a computer, and analyzed using an OSMA software program. Further experimental details are provided elsewhere (Einarsdbttir, ‘O., Dawes, T.D. and Georgiadis, K., unpublished results).
Results and Discussion.
Figure 1A shows the ground state near-infrared
absorption
spectra of the fully reduced unliganded and the fully reduced CO complexes of cytochrome oxidase.
Two absorption bands at 785 nm (E -50 M-lcm-1)
and 710 nm (E -80 M-lcm-1)
are
clearly observed in the fully reduced unliganded enzyme. Figure 2A shows the baseline-corrected spectra of Figure 1A. Neither band has been reported previously in the ground state absorption spectra of cytochrome oxidase. The 785 nm band, if present in the spectra of the fully oxidized enzyme and the mixed-valence CO complex, would be masked by the much more intense 830 nm band (l-3). As pointed out below, we do not expect the 785 nm band to be present in the absorbance spectra of these complexes. Figures 1A and 2A show that the 785 nm band disappears when CO binds to cytochrome 3. Therefore, this absorbance is attributed to the five coordinate high-spin unliganded cytochrome a32+. This is supported by time-resolved flash-photolysis studies of the fully reduced and the mixed-valence CO complexes (vi& infru). The 7 10 nm band is present in both the fully reduced unliganded and the fully reduced CO enzyme (Figs. 1A and 2A). This band appears to be slightly blue-shifted, -2 nm, in the fully reduced CO enzyme (Fig. 2A), which accounts for a small increase at -720 nm in the reduced minus CO difference spectrum (Fig. 1B). 1036
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Figure 1. (A) Ground state near-infrared absorption spectra of fully reduced unliganded (solid line) and fully reduced CO (broken line)cytochrome Eoxidase.The cytochromeoxidasein 0.1 M pH 7.4 Na-phosphate buffer was -0.4 mM in total heme. Both spectra have been downshifted
0.08absorbance units. (B) ReducedminusCOdifferencespectrum. Figure 2. (A) Baseline-corrected spectraof Figure 1A. A multiple point polynomialwas subtractedfrom the slopingbackgroundof the spectrain Figure 1A. Solid line: Fully reduced cytochromeoxidase.Brokenline: Fully reducedCO-boundcytcchrome oxidase. (B) Baselinecorrected time-resolvedspectraobtained 10 ns (solid line) and 10 ps (broken line) after photodissociation of CO from thefully reducedenzyme.The spectrahavebeennormalizedto the concentrationof the samplein Figure 1 andto 100%photolysis.Original spectraareshownin Figure3A.
Figure 3A showsthe time-resolveddifference spectraobtainedfollowing photodissociation of CO from cytochrome a2+ in the fully reducedenzyme. The absorbancechangeis the difference in the absorbancemeasuredat the indicated times after the laserflash and before the flash. Figure 2B showsthe baseline-correctedspectraobtained 10 ns and 10 ps after photolysis. Thesespectra have beennormalized to the concentrationof the samplein Figure 1 and to 100%photolysis (from -60%). The appearanceof an absorbanceat 785 nm in the 10 ns spectrum indicates that the unligandedcytochrome a2+ photoproduct is five-coordinate and high spin asin the fully reduced unliganded enzyme. Appearance of a band at 780 nm was reported previously by Boelens and 1037
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Figure 3. (A) Time-resolved near-infrared absorption difference spectra obtained following photodissociation of CO from fully reduced cytochrome s oxidase. Cytochrome oxidase concentration was -320 t.tM in total heme and the spectra were obtained along the 1 cm pathlength. The spectra represent the absorbance after photolysis minus before photolysis. The spectra are an average of 2.56 single spectra. Spectra were recorded at the following times after CO photolysis: a) 10 ns, b) 10 J~S,c) 100 ps, d) 3 ms, e) 7 ms, f) 12 ms, and g) 30 ms. (B) Time-resolved nearinfrared absorption difference spectra obtained following photodissociation of CO from mixedvalence cytochrome c oxidase. Cytochrome oxidase concentration was -300 PM in total heme. Spectra were recorded at the following times after CO photolysis : a) 10 ns, b) 10 t~s, c) 50 t.t’s,d) 500 ps, e) 7 ms, and f) 30 ms.
Wever (12) in the 1 ms difference spectrum of the photolyzed fully reduced CO cytochrome oxidase. The 785 nm band disappearswith a half-life of -7 msec (Fig. 3A). This rate of disappearanceis the sameasthe rate of recombinationof CO with cytochrome a2+ asmeasuredat 445 nm (13), providing further support for the assignmentof this band to the ferrous unliganded cytochrome a. The 785 nm band is also observedfollowing photolysis of CO from cytochrome & in the mixed-valence enzyme (Fig. 3B). In this complex, the 785 nm band appearsto decreaseon the sametimescaleas in the fully reduced CO-photolyzed enzyme, although this is more difficult to evaluate due to a simultaneouselectron transfer amongthe metal centersin this complex (12). As evident from Figure 3B, an absorbancedecreaseis observedat 830 nm, which occurs with a half1038
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life of -50-100 i.ts. This decrease is attributed to 5-10% of CUA~+ becoming reduced following photolysis of CO from the mixed-valence enzyme as reported earlier (12,14,15>. The decrease in the 830 nm band is more pronounced following photolysis of CO from the three electron reduced oxidase (not shown), in accordance with previous studies (1516).
A decrease in the 785 nm band
due to 5-10% oxidation of cytochrome a, suggested to occur on microsecond timescale following photolysis of CO from the mixed-valence enzyme (12,14,15), would be too small to detect within the signal to noise ratio of Figure 3B. The 785 nm band, except for its rather low extinction coefficient, signatures and characteristics
as band III in deoxy-hemoglobin
has similar spectral
and deoxy-myoglobin
(4,5). The
bands are similar in energy and bandwidth (7,8) and are both spin state sensitive, i.e., disappear on going from the unliganded five coordinate high-spin forms to the six coordinate low-spin complexes.
CO
Therefore, the 785 nm band in cytochrome oxidase may be the heme a analogue of
band III in deoxy-hemoglobin
and deoxy-myoglobin,
785 nm band could represent a d+d
but with lesser intensity. Alternatively,
transition of the unliganded cytochrome
the
a32+. Circular
dichroism and magnetic circular dichroism measurements which may aid in further assignment of this band are in progress. Band III of photodissociated myoglobin is unshifted relative to that of deoxy-myoglobin within 10 ns of photolysis, while band III of photodissociated hemoglobin is red-shifted by -6 nm from its equilibrium
deoxy position (7). At 100 ns it has relaxed about half-way
to the deoxy-
hemoglobin value and changes further to about 100 ps. These results have been correlated with results from transient optical studies which have shown that myoglobin relaxes to its equilibrium deoxy position within 30 ps of CO photolysis, hemoglobin
whereas the heme pocket of photodissociated
relaxes on much longer timescale (17,18).
conformationally
Band III has been suggested to be a
sensitive indicator of the heme-pocket geometry in hemoglobin and in Fe-Mn
hybrid hemoglobins through the Fe-proximal
histidine bond (7,8). However,
observed in the 10 ns unliganded CO photoproducts
the 785 nm band
of the fully reduced and the mixed-valence
cytochrome oxidase complexes is unshifted in wavelength and unchanged in intensity relative to the equilibrium fully reduced cytochrome oxidase (Fig. 2) within the signal to noise ratio of these spectra.
There may be a slight blue shift almost within the signal to noise ratio in the 10 ~.rs
spectrum (Fig. 2B). We have previously reported a significant absorbance decrease in the cx band on a microsecond timescale (tI/2 -1.5 ps) following photolysis of CO from fully reduced cytochrome oxidase (19). We attributed this decrease to an axial ligand perturbation of the cytochrome &-j triggered by the loss of CO from CUB+-CO into solution as demonstrated by timeresolved infrared spectroscopy (19,20). Therefore, the 785 nm band in cytochrome oxidase does not appear to be as sensitive to axial ligand perturbation as band III in deoxy-hemoglobin. The 710 nm band is present both in the fully reduced and in the fully reduced CO-bound enzyme (Fig. 1) but is absent in the fully oxidized and the mixed-valence CO-bound complexes. Upon reducing the enzyme past the two electron reduced state by prolonged incubation under CO, the 710 nm band appears and becomes maximum for the fully reduced CO-bound enzyme. This band may be a d+d transition of the low spin ferrous cytochrome $+. Ferrous cytochrome c with axial ligands histidine and methionine has been reported to have low intensity near-infrared 1039
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absorption bands at 630,730 and 8 16 nm (E -200 M-‘cm-l) experiments, cytochrome
these transitions
E. The absence of similar transitions
carboxy-myoglobin methionine,
have been assigned to d+d
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(21,22). Based on circular dichroism transitions
of the low spin ferrous
in the spectra of carboxy-hemoglobin
and
has been attributed to a larger ligand field strength of CO compared to
causing these transitions
to shift to higher energy and be buried by the intense
porphyrin X+X* transitions (4,21,22). This suggests that the low spin ferrous cytochrome a with two axial histidines
has a weaker
ligand field than the carboxy-hemoglobin
and carboxy-
myoglobin, causing the 7 10 nm band to be observed in cytochrome oxidase. As mentioned above, there appears to be a small blue shift in the 710 nm band upon CO binding to the fully reduced enzyme (Figs. 1B and 2A). This suggests that coordination of CO to cytochrome ~3~+ may affect the electronic structure of cytochrome a. Recent second derivative spectroscopic studies on cytochrome oxidase have suggested that ferrous cytochrome 1 may have multiple conformational states depending on the ligation state of cytochrome 8j2+ (23). In conclusion, these results represent the first demonstration of near-infrared
absorption
bands in the spectra of fully reduced cytochrome oxidase, fully reduced CO-bound oxidase, and photoproducts of CO-photodissociated cytochrome oxidase complexes. Since only one of the cytochromes contributes to each band, the 785 nm and the 7 10 nm bands provide unique markers for the ferrous five coordinate high-spin cytochrome @ 2+ and the six coordinate low-spin ferrous cytochrome a2+, respectively.
Acknowledgment.
Acknowledgement
is made to the donors of The Petroleum Research Fund,
administered by the ACS, for partial support of this research.
References :. 3: 4. i: 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Boelens, R. and Wever, R. (1980) FEBS Lett. 116,223-226. Wharton D.C. and Tzagoloff, A. (1964) J. Biol. Chem. m, 2036-2041. Beinert, H., Shaw, R-W., Hansen, R.E. and Hartzell, C.R. (1980) Biochim. Biphys. Acta m, 458-470. Eaton, W.A., Hanson, LX., Stephens, P.J., Sutherland, J.C. and Dunn J.B.R. (1978) J. Am. Chem. Sot. yM, 4991-5003. Eaton, W.A. and Hofrichter, J. (1981) Methods Enzymol. 26, 175-261. Ansari A., Berendzen, J., Bowne, S.F., Frauenfelder, H., Iben, I.E.T., Sauke, T.B., Shyamsunder, E. and Young, R.D. (1985) Proc. Natl. Acad. Sci. U.S.A. &&5ooO-5004. Sassaroli, M. and Rousseau, D.L. (1987) Biochemistry 26,3092-3098. Chavez, M.D., Courtney, S.H., Chance, M.R., Kuila, D., Nocek, J., Hoffman B.M., Friedman, J.M. and Ondrias, M.R. (1990) Biochemistry 2p, 4844-4852. Georgiadis, K., Dawes, T. and Einardbttir, ‘0. (1992) Biophysical J. d, 1158. Yoshikawa, S., Choc, M.G., O’Toole, M.C. and Caughey, W.S. (1977) J. Biol. Chem. 252, 5498-5508. Goldbeck, R.A., Dawes, T.D., Milder, S.J., Lewis, J.W. and Kliger, D.S. (1989) Chem. Phys. Lett. m, 545-549. Boelens R., Wever, R. and Van Gelder, B.F. (1982) B&him. Biophys. Acta m, 264-272. Gibson, Q.H. and Greenwood, C. (1963) Biochem J. &, 541-554. Brzezinski, P. and Malmstrom B.G. (1987) Biochem. Biophys. Acta &@,29-38. Oliveberg, M. and Malmstrom B.G. (1991) Biochemistry Xl, 7053-7057. Morgan, J.E., Li, P.M., Jang, D.-J., El-Sayed, M.A. and Chan, S.I. (1989) Biochemistry 2, 6975-6983. 1040
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17. Findsen E.W., Scott, T.W., Chance, M.R., Friedman, J.M. and Ondrias, M.R. (1985) J. Am. Chem. Sot. m, 33553357. 18. ~4~;~~.M., Dalickas, G.A., Eaton, W.A. and Hochstrasser, R.M. (1988) Biophys. J. a, 19. Woodruff, W-H., Einarsdottir, ‘O., Dyer, R.B., Bagley, K.A., Palmer, G., Atherton, S.J., Goldbeck, R.A., Dawes, T.D. and Kliger, D.S. (1991) Proc. Natl. Acad. Sci. U.S.A. &$, 2588-2592. 20. Dyer, R.B., Einarsdottir, ‘O., Killough, P.M., Lopez-Garriga, J.J. and Woodruff, W.H. (1989) J. Am. Chem. Sot. u,7657-7659. 21. Eaton, W.A. and Chamey, E. (1969) J. Chem. Phys. a, 4502-4505. 22. Eaton, W.A. and Chamey, E. (1971) in Probes of Structure and Function of Macromolecules UMembraneS, Vol. I, (Chance, B., Lee, C.P. and Blasie, J.K., eds) pp. 155-164, Academic Press, New York. 23. Sherman, D., Kotake, S., Ishibe, N. and Copeland, R.A. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4265-4269.
1041
Vol. 184, No. 2, 1992 April 30, 1992
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1035-1041
EVIDENCE FOR A BAND III ANALOGUE IN THE NEAR-INFRARED ABSORPTION SPECTRA OF CYTOCHROME E OXIDASE ‘Olaf Einarsdktir, Katy E. Georgiadis,and Timothy D. Dawes Departmentof Chemistry, University of California, SantaCruz SantaCruz, California 95064 Received
March
17,
1992
Summary. Ground state near-infrared absorption spectraof fully reduced unliganded and fully reducedCO @+ CUA+g2+-CO CUB+)cymchrome Eoxidasewere investigated. Flash-photolysis time-resolved absorptiondifference spectraof the mixed-valence h3+ CUA~+a32+-C0 CUB+)and the fully reduced CO complexes were also studied. A band near 785 nm (E -50 M-km-l) was observed in the fully reducedunliganded enzyme and the CO photoproducts. The time-resolved 785 nm banddisappearedon the sametimescale(tin -7 ms) asCO recombinedwith cytochrome a32+. This band, which is attributed to the unligandedfive coordinate ferrous cytochrome a2+, has somecharacteristicsof band III in deoxy-hemoglobin and deoxy-myoglobin. A secondband was observed at -710 nm (E -80 M-km-l) in the fully reducedunligandedand the fully reduced CO complexes. This band, which we assignto the low spin ferrous cytochrome 3, appearsto be affected by the ligation stateat the cytochromea32+site. 0 1992 AcademicPress, 1°C.
Optical absorptionstudieson cytochromeoxidasein the near-infraredregion have primarily concentrated on the fully oxidized enzyme, which showsa broad band at 830 nm. This band, which is also present in the mixed-valence CO complex (a3+ CUA~+aj2+-CO CUB+), has been attributed to CUA~+(l-3). The fully reducedunligandedand the fully reduced CO-bound enzyme have no reported absorbancesin this region. However, five coordinate high spin deoxyhemoglobin and deoxy-myoglobin have been shownto have a charge-transfertransition near 760 nm with E -200 M-km-l
(4,5). This transition, designatedband III, has also been observed
following photodissociationof CO from theseproteins (6-8). This bandis absentin the spectraof the six coordinate CO-bound proteins, which show no absorbancesin this region. Based on circular dichroism, magnetic circular dichroism, and polarized single-crystal absorption,Eaton & al. (4) have assignedthis transition to a charge-transfertransition betweenthe porphyrin x system and the iron K system(azu + dzy). Until recently, similar absorbanceshad not beenreported for cytochrome E oxidase (9). In this paper, we report the presenceof a bandat -785 nm in the near infrared spectrumof fully reducedunligandedcytcchrome Eoxidaseand in the time-resolveddifference spectraobtained following photodissociationof CO from the mixed-valence and the fully reducedcomplexes. This band is attributed to the unliganded five coordinate ferrous cytochrome a32+. A secondband is 0006-291X/91$1.50 1035
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observed near -7 10 nm in the ground state infrared spectra of the fully reduced unliganded and the fully reduced CO-bound enzyme. We assign this band to the ferrous low-spin cytochrome a.
mrials and Metho& Cytochmme oxidase was isolated according to the procedure of Yoshikawa ti d. (10). prior to reduction, the oxidized enzyme in 0.1 M Na-phosphate pH 7.4 was filtered through a 0.2 pm filter into a fluorescence sample cuvette (10x4 mm) equipped with a stopcock for enzyme deoxygenation. The deoxygenated enzyme was reduced under N2 with a small excess of sodium dithionite. The fully reduced CO complex was formed by blowing CO over the enzyme solution for 15-30 minutes. The two electron reduced mixed-valence Co complex was obtained by incubating the deoxygenated oxidized enzyme under 1 atm CO for several hours. The CO complexes were characterized by their visible spectra which were recorded before and after the photolysis measurements to ensure that the samples did not change during the measurements. The ground state near-infrared spectra were recorded on an IBM 9420 UV-Vis spectrophotometer. Cytochrome oxidase concentration was determined using an extinction coefficient of the oxidized enzyme at 598, 8.5 mM-lcm-l in total heme, or an absorbance coefficient of 2.3 mM-Icm-1 (reduced minus oxidized) at 830 nm for CuA2+ (1). The extinction coefficients of the near-infrared bands were determined from the baseline-corrected spectra, which were obtained by a subtraction of a multiple point polynomial fit from the sloping absorbance background. The CO-bound complexes were photolyzed using a DCR-11 Nd:YAG laser (532 nm, 80 rnI/pulse, 7ns duration) with a repetition rate of 2 Hz. The photolysis laser beam was partially focused with a cylindrical lens along the 4 mm path and 90” to the probe beam. The probe beam, a high-power Xenon flashlamp of pulse width 2 lrs (1 l), was focused on the sample cell along the 10 mm path and then recollimated and focused on the entrance slit of a spectrograph/ monochromator. The time-resolved near-infrared spectra of the CO-photodissociated enzyme derivatives were recorded at many wavelengths simultaneously using an optical spectrometric multichannel analyzer (OSMA, Princeton Instruments, Inc.) detector. The spectra at various times following photolysis were obtained by varying the time of an intensifier gate pulse (gate width 5 ns to 2.5 ps) relative to the excitation pulse using a delay generator. The spectra were averaged, transferred to a computer, and analyzed using an OSMA software program. Further experimental details are provided elsewhere (Einarsdbttir, ‘O., Dawes, T.D. and Georgiadis, K., unpublished results).
Results and Discussion.
Figure 1A shows the ground state near-infrared
absorption
spectra of the fully reduced unliganded and the fully reduced CO complexes of cytochrome oxidase.
Two absorption bands at 785 nm (E -50 M-lcm-1)
and 710 nm (E -80 M-lcm-1)
are
clearly observed in the fully reduced unliganded enzyme. Figure 2A shows the baseline-corrected spectra of Figure 1A. Neither band has been reported previously in the ground state absorption spectra of cytochrome oxidase. The 785 nm band, if present in the spectra of the fully oxidized enzyme and the mixed-valence CO complex, would be masked by the much more intense 830 nm band (l-3). As pointed out below, we do not expect the 785 nm band to be present in the absorbance spectra of these complexes. Figures 1A and 2A show that the 785 nm band disappears when CO binds to cytochrome 3. Therefore, this absorbance is attributed to the five coordinate high-spin unliganded cytochrome a32+. This is supported by time-resolved flash-photolysis studies of the fully reduced and the mixed-valence CO complexes (vi& infru). The 7 10 nm band is present in both the fully reduced unliganded and the fully reduced CO enzyme (Figs. 1A and 2A). This band appears to be slightly blue-shifted, -2 nm, in the fully reduced CO enzyme (Fig. 2A), which accounts for a small increase at -720 nm in the reduced minus CO difference spectrum (Fig. 1B). 1036
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Figure 1. (A) Ground state near-infrared absorption spectra of fully reduced unliganded (solid line) and fully reduced CO (broken line)cytochrome Eoxidase.The cytochromeoxidasein 0.1 M pH 7.4 Na-phosphate buffer was -0.4 mM in total heme. Both spectra have been downshifted
0.08absorbance units. (B) ReducedminusCOdifferencespectrum. Figure 2. (A) Baseline-corrected spectraof Figure 1A. A multiple point polynomialwas subtractedfrom the slopingbackgroundof the spectrain Figure 1A. Solid line: Fully reduced cytochromeoxidase.Brokenline: Fully reducedCO-boundcytcchrome oxidase. (B) Baselinecorrected time-resolvedspectraobtained 10 ns (solid line) and 10 ps (broken line) after photodissociation of CO from thefully reducedenzyme.The spectrahavebeennormalizedto the concentrationof the samplein Figure 1 andto 100%photolysis.Original spectraareshownin Figure3A.
Figure 3A showsthe time-resolveddifference spectraobtainedfollowing photodissociation of CO from cytochrome a2+ in the fully reducedenzyme. The absorbancechangeis the difference in the absorbancemeasuredat the indicated times after the laserflash and before the flash. Figure 2B showsthe baseline-correctedspectraobtained 10 ns and 10 ps after photolysis. Thesespectra have beennormalized to the concentrationof the samplein Figure 1 and to 100%photolysis (from -60%). The appearanceof an absorbanceat 785 nm in the 10 ns spectrum indicates that the unligandedcytochrome a2+ photoproduct is five-coordinate and high spin asin the fully reduced unliganded enzyme. Appearance of a band at 780 nm was reported previously by Boelens and 1037
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b-m)
Figure 3. (A) Time-resolved near-infrared absorption difference spectra obtained following photodissociation of CO from fully reduced cytochrome s oxidase. Cytochrome oxidase concentration was -320 t.tM in total heme and the spectra were obtained along the 1 cm pathlength. The spectra represent the absorbance after photolysis minus before photolysis. The spectra are an average of 2.56 single spectra. Spectra were recorded at the following times after CO photolysis: a) 10 ns, b) 10 J~S,c) 100 ps, d) 3 ms, e) 7 ms, f) 12 ms, and g) 30 ms. (B) Time-resolved nearinfrared absorption difference spectra obtained following photodissociation of CO from mixedvalence cytochrome c oxidase. Cytochrome oxidase concentration was -300 PM in total heme. Spectra were recorded at the following times after CO photolysis : a) 10 ns, b) 10 t~s, c) 50 t.t’s,d) 500 ps, e) 7 ms, and f) 30 ms.
Wever (12) in the 1 ms difference spectrum of the photolyzed fully reduced CO cytochrome oxidase. The 785 nm band disappearswith a half-life of -7 msec (Fig. 3A). This rate of disappearanceis the sameasthe rate of recombinationof CO with cytochrome a2+ asmeasuredat 445 nm (13), providing further support for the assignmentof this band to the ferrous unliganded cytochrome a. The 785 nm band is also observedfollowing photolysis of CO from cytochrome & in the mixed-valence enzyme (Fig. 3B). In this complex, the 785 nm band appearsto decreaseon the sametimescaleas in the fully reduced CO-photolyzed enzyme, although this is more difficult to evaluate due to a simultaneouselectron transfer amongthe metal centersin this complex (12). As evident from Figure 3B, an absorbancedecreaseis observedat 830 nm, which occurs with a half1038
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life of -50-100 i.ts. This decrease is attributed to 5-10% of CUA~+ becoming reduced following photolysis of CO from the mixed-valence enzyme as reported earlier (12,14,15>. The decrease in the 830 nm band is more pronounced following photolysis of CO from the three electron reduced oxidase (not shown), in accordance with previous studies (1516).
A decrease in the 785 nm band
due to 5-10% oxidation of cytochrome a, suggested to occur on microsecond timescale following photolysis of CO from the mixed-valence enzyme (12,14,15), would be too small to detect within the signal to noise ratio of Figure 3B. The 785 nm band, except for its rather low extinction coefficient, signatures and characteristics
as band III in deoxy-hemoglobin
has similar spectral
and deoxy-myoglobin
(4,5). The
bands are similar in energy and bandwidth (7,8) and are both spin state sensitive, i.e., disappear on going from the unliganded five coordinate high-spin forms to the six coordinate low-spin complexes.
CO
Therefore, the 785 nm band in cytochrome oxidase may be the heme a analogue of
band III in deoxy-hemoglobin
and deoxy-myoglobin,
785 nm band could represent a d+d
but with lesser intensity. Alternatively,
transition of the unliganded cytochrome
the
a32+. Circular
dichroism and magnetic circular dichroism measurements which may aid in further assignment of this band are in progress. Band III of photodissociated myoglobin is unshifted relative to that of deoxy-myoglobin within 10 ns of photolysis, while band III of photodissociated hemoglobin is red-shifted by -6 nm from its equilibrium
deoxy position (7). At 100 ns it has relaxed about half-way
to the deoxy-
hemoglobin value and changes further to about 100 ps. These results have been correlated with results from transient optical studies which have shown that myoglobin relaxes to its equilibrium deoxy position within 30 ps of CO photolysis, hemoglobin
whereas the heme pocket of photodissociated
relaxes on much longer timescale (17,18).
conformationally
Band III has been suggested to be a
sensitive indicator of the heme-pocket geometry in hemoglobin and in Fe-Mn
hybrid hemoglobins through the Fe-proximal
histidine bond (7,8). However,
observed in the 10 ns unliganded CO photoproducts
the 785 nm band
of the fully reduced and the mixed-valence
cytochrome oxidase complexes is unshifted in wavelength and unchanged in intensity relative to the equilibrium fully reduced cytochrome oxidase (Fig. 2) within the signal to noise ratio of these spectra.
There may be a slight blue shift almost within the signal to noise ratio in the 10 ~.rs
spectrum (Fig. 2B). We have previously reported a significant absorbance decrease in the cx band on a microsecond timescale (tI/2 -1.5 ps) following photolysis of CO from fully reduced cytochrome oxidase (19). We attributed this decrease to an axial ligand perturbation of the cytochrome &-j triggered by the loss of CO from CUB+-CO into solution as demonstrated by timeresolved infrared spectroscopy (19,20). Therefore, the 785 nm band in cytochrome oxidase does not appear to be as sensitive to axial ligand perturbation as band III in deoxy-hemoglobin. The 710 nm band is present both in the fully reduced and in the fully reduced CO-bound enzyme (Fig. 1) but is absent in the fully oxidized and the mixed-valence CO-bound complexes. Upon reducing the enzyme past the two electron reduced state by prolonged incubation under CO, the 710 nm band appears and becomes maximum for the fully reduced CO-bound enzyme. This band may be a d+d transition of the low spin ferrous cytochrome $+. Ferrous cytochrome c with axial ligands histidine and methionine has been reported to have low intensity near-infrared 1039
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absorption bands at 630,730 and 8 16 nm (E -200 M-‘cm-l) experiments, cytochrome
these transitions
E. The absence of similar transitions
carboxy-myoglobin methionine,
have been assigned to d+d
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(21,22). Based on circular dichroism transitions
of the low spin ferrous
in the spectra of carboxy-hemoglobin
and
has been attributed to a larger ligand field strength of CO compared to
causing these transitions
to shift to higher energy and be buried by the intense
porphyrin X+X* transitions (4,21,22). This suggests that the low spin ferrous cytochrome a with two axial histidines
has a weaker
ligand field than the carboxy-hemoglobin
and carboxy-
myoglobin, causing the 7 10 nm band to be observed in cytochrome oxidase. As mentioned above, there appears to be a small blue shift in the 710 nm band upon CO binding to the fully reduced enzyme (Figs. 1B and 2A). This suggests that coordination of CO to cytochrome ~3~+ may affect the electronic structure of cytochrome a. Recent second derivative spectroscopic studies on cytochrome oxidase have suggested that ferrous cytochrome 1 may have multiple conformational states depending on the ligation state of cytochrome 8j2+ (23). In conclusion, these results represent the first demonstration of near-infrared
absorption
bands in the spectra of fully reduced cytochrome oxidase, fully reduced CO-bound oxidase, and photoproducts of CO-photodissociated cytochrome oxidase complexes. Since only one of the cytochromes contributes to each band, the 785 nm and the 7 10 nm bands provide unique markers for the ferrous five coordinate high-spin cytochrome @ 2+ and the six coordinate low-spin ferrous cytochrome a2+, respectively.
Acknowledgment.
Acknowledgement
is made to the donors of The Petroleum Research Fund,
administered by the ACS, for partial support of this research.
References :. 3: 4. i: 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
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