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The reactivity of pulsed cytochrome c oxidase toward carbon monoxide
The Reactivity of Pulsed Cytochrome c Oxidase Toward Carbon Monoxide Joel E. Morgan, David F. Blair, and Sunney I. Chan Arthur Amos Noyes Laboratory of Chemical Physics, California Insitute of Technology
ABSTRACT When pulsed cytochrome c oxidase is exposed to carbon monoxide in the absence of oxygen the enzyme is converted quickly to its CO-associated mixed valence state. The half-time for this reaction at 0°C is about 4 min. This is about 100 times faster than a similar reaction which begins with the resting form of the enzyme. The possible significance of this reaction in understanding the pulsed/resting phenomenon and the carbon monoxide oxygenase reactions of cytochrome oxidase is discussed.
INTRODUCTION Cytochrome c oxidase is the terminal enzyme in the mitochondrial electron transfer chain; here the respiratory electrons are finally used to reduce dioxygen to water. The enzyme receives four equivalents of electrons from cytochrome c, reduces dioxygen, and conserves the energy from this reaction as a pH gradient across the inner mitochondrial membrane, in part via the uptake of protons in the reduction of oxygen to water, and in part by the electrogenic pumping of protons from the mitochondrial matrix to the cytosol. (For recent reviews on cytochrome oxidase see Refs. l-3.) To accomplish these tasks cytochrome oxidase utilizes four different metal centers: two iron hemes, with the unusual heme A porphyrin, and two coppers. One iron and one copper ion, namely, cytochrome a and copper A, are buried in the protein and do not react with most externally added ligands. These two low potential centers transfer electrons from cytochrome c to the oxygen binding site, and it has been suggested that one of these sites is the primary locus of the proton pumping function. The other two metal centers, cytochrome a3 and copper B, are close together and make up the oxygen binding/reduction site. They are accessible to a number of exogenous ligands in addition to molecular oxygen [3]. Address reprint requests to Dr. Sunney I. Chan, Arthur Amos Noyes Laboratory Institute
of Technology,
Pasadena,
Journal of Inorganic Biochemistry 23, 295-302 (1985) 0 1985 Elsevier Science Publishing Co., Inc. 52 Vanderbilt
of Chemical
Physics,
California
CA 91125.
Ave., New York, NY 10017
295 0162-0134/85/$3.30
296
J. E. Morgan, D. F. Blair and S. I. Chan
Oxidized cytochrome oxidase exhibits at least two states which have different activity. When isolated from the mitochondrion, the enzyme is in a relatively state. Reduction and reoxidation transform the enzyme inactive “resting” temporarily into an activated state known as “pulsed” or “oxygen-pulsed” [4, 51. Both pulsed and resting forms of the oxidized enzyme have the same overall oxidation state, but the pulsed enzyme can be reduced more quickly and reaches steady state turnover sooner than the resting enzyme [5, 61. The pulsed enzyme owes its enhanced activity to faster intramolecular electron transfer between the low potential centers (cytochrome a, copper A) and the oxygen binding site [5]. If the enzyme is not continuously rereduced it decays back to the resting state within a few hours. The pulsed state of the enzyme is also characterized by distinctive features in the visible absorption spectrum which are probably due to cytochrome a3, and which indicate that the oxygen binding site metal centers are in a different ligation state [7, 81. One of the most useful probes in studying the structure of the oxygen binding site is carbon monoxide (CO) [9]. Carbon monoxide binds to reduced cytochrome a3 and stabilizes both cytochrome a3 and copper B in their reduced states. In the carbon monoxide complex, cytochrome a and copper A can either be reduced or oxidized. When both low potential sites are oxidized, the half-reduced state is known as the CO-mixed-valence compound. Since this compound is both photosensitive and reactive with oxygen it has been used as the starting point for a number of kinetic studies 110, 1 I]. The CO-mixed-valence compound has usually been prepared by partial reoxidation of the fully reduced, CO-inhibited enzyme [ 121, but it can be made directly from the resting form of the enzyme by incubating the sample for many hours under carbon monoxide in the absence of oxygen. Evidence has recently been presented [13] that in the latter case the enzyme is being reduced by the carbon monoxide itself in a water-gas shift reaction of the form FeUj3+ +3CO+H,O+ clluz+
Fe,s2+-CO CUB’+
+ CO2 + 2H+
We have found that if carbon monoxide is added to the pulsed rather than to the resting enzyme, the CO-mixed-valence compound is formed in minutes instead of hours. This dramatic difference in the reactivity of the pulsed vs. the resting enzyme towards carbon monoxide is the subject of the present study. We will briefly discuss the manner in which this result might contribute to our understanding of both the pulsed/resting phenomenon and the carbon monoxide oxygenase reactions in which cytochrome oxidase reportedly catalyzes the oxidation of carbon monoxide to carbon dioxide by molecular oxygen [14, 151.
EXPERIMENTAL Beef heart cytochrome c oxidase was prepared by the method of Hartzell and Beinert [16]. Enzyme samples were diluted in a buffer of 50 mM tris and 0.5% Tween-20 (w/v), at pH 7.4. Enzyme concentrations were determined using an extinction coefficient of 39.6/mM/cm at 605 nm 1171. All experiments were performed at ice temperature.
Pulsed Cytochrome
c Oxidase
297
Enzyme samples were made anaerobic by replacing the atmosphere in the cuvette with argon and then agitating to equilibrate the liquid with the atmosphere. Five such cycles were usually used. Argon used for the anaerobic work was made free of oxygen by means of a l-m-long scrubbing column containing manganese dioxide supported on vermiculite. Carbon monoxide (99.99% pure) was obtained from Matheson Co. In preparing the reduced enzyme, NADH was used as the reductant with phenazine methosulfate (PMS) [ 181 as the electron mediator. Both were obtained from Sigma. Reductant solutions were prepared with 1.65 mM NADH and 16.1 PM PMS in 50 mM Hepes buffer pH 7.4. NADH concentrations were determined using a millimolar extinction coefficient of 6220 at 338 nm [19]. The absorbance of the mediator was ignored. Visible absorption spectra were recorded using a Beckman Acta CIII spectrometer interfaced to a Spex Industries SCAMP SC-31 data processor. Spectra were stored on magnetic (floppy) disk. Spectral bandwidth was 1 nm, except for NADH assays when 2 nm was used. Wavelength calibration was carried out with a holomium oxide calibration filter. Preparation of the Pulsed Enzyme Samples of pulsed enzyme were prepared by reducing the enzyme fully (four electrons) and then reoxidizing with oxygen. In preparing the reduced enzyme for this purpose, a great deal of care was taken to use as close as reasonably possible to the exact amount of reductant necessary to reduce the sample fully. If too much reductant is used there may be some left over after reoxidation to rereduce the sample. Since reduced cytochrome oxidase binds carbon monoxide rapidly, this could produce misleading results concerning possible reactions between the pulsed form of the enzyme and carbon monoxide. Too little reductant, on the other hand, would leave some enzyme molecules incompletely reduced and thus produce species other than the pulsed enzyme, containing partly reduced oxygen intermediates. These other species could have very different reactivities towards carbon monoxide from the four-electron pulsed species. In order to achieve precise fourelectron reduction, both the enzyme sample and the reductant solution were first thoroughly deaerated. The reductant solution was then assayed spectrophotometritally and the calculated amount of reductant transferred anaerobically by syringe to the sample cuvette. The enzyme sample was kept at ice temperature until reduction was complete. The reductant assay was necessary because the NADH/ PMS solution is air sensitive and the concentration of reductant may change if all of the air is not removed Our experiments with carbon monoxide required oxygen-pulsed cytochrome oxidase samples which were free of excess oxygen. This was achieved either by adding a very small (close to stoichiometric) amount of oxygen dissolved in water, or by opening the cell to air and then removing the excess oxygen by washing with argon as described above. RESULTS Oxidized cytochrome oxidase is converted into the CO-mixed-valence
compound
298
J. E. Morgan, D. F. Blair and S. I. Chan
when it is incubated under carbon monoxide in the absence of oxygen, This happens whether the enzyme is in its resting or pulsed state (Fig. 1). However, the product appears much more rapidly starting from the pulsed enzyme. Figure 2 shows the time course of these reactions as followed by visible spectroscopy. The half times are about 400 min for the resting enzyme (traces A and B) and about 4 min for the pulsed enzyme (trace C). This unusual reactivity of the pulsed enzyme toward carbon monoxide is lost slowly over the course of several hours. When carbon monoxide is introduced up to 45 min after reoxidation (trace C) there appears to have been little loss of activity, since almost all of the sample is converted quickly to the CO-mixedvalence product. However, if 220 min elapse between reoxidation and addition of carbon monoxide (trace D), the sample behaves like a mixture of pulsed and resting enzyme; that is, the reaction takes place with both a fast phase and a slow phase. If the delay is lengthened to 360 minutes (trace E), the enzyme appears to have returned almost completely to the resting state. It should be noted that there appears to be a small fast component even in the reactions starting with the resting form of the enzyme (traces A and B). The pulsed enzyme used in these experiments was prepared by reducing the resting enzyme with a stoichiometric amount of reductant and then reoxidizing with oxygen. The objective was to produce a large population of the fully oxidized pulsed species in the absence of any excess reductant which might rereduce the enzyme. Avoiding excess reductant is important since reduced cytochrome oxidase binds carbon monoxide very quickly; in the present experiment, if the enzyme was being reduced from some other source, it might still appear that the carbon monoxide was responsible. Control experiments showed that this problem was avoided: When enzyme samples were deliberately reduced with an excess of reductant the resulting pulsed enzyme quickly became fully rereduced (four electrons) in the presence of carbon monoxide; however, when no excess was used, the fast rereduction stopped at the two electron (CO-mixed-valence) stage. It should also be noted that although the samples for the carbon monoxide reaction must be free of oxygen, the pulsed enzyme was produced by aerobic reoxidation; thus, either a very small (close to stoichiometric) amount of oxygen was used, or an excess of oxygen was added and then removed before the addition of carbon monoxide. If some oxygen was still present when carbon monoxide was added to the pulsed enzyme, the CO-mixed-valence compound was not seen immediately. Instead, the spectrum of a species known as the “607-nm complex” [14] was observed. The “607-nm complex” is the species produced when the CO-mixed-valence compound reacts with dioxygen and is so named because it has a peak at 607 nm when compared to the resting enzyme. The “607-nm complex” persisted for some time in the visible spectrum and then, presumably when the oxygen was exhausted, the CO-mixed-valence compound appeared quickly.
DISCUSSION We have shown that in the absence of oxygen, carbon monoxide rapidly converts pulsed cytochrome oxidase into the CO-mixed-valence compound, a process which involves both reduction by two electrons and binding of carbon monoxide to
Msed
Cytochrome c Oxidase
700
299
600
500 WAVELENGTH
4co
J
(nm)
FIGURE 1.
(a) Visible absorbance spectra of cytochrome ox&se: (i) . . . , resting; (ii) -, pulsed; (iii) - - -, Co-mixed valence compound. All spectra normalized to cytochrome oxidase concentration 4.6 CM, path length 10 mm. (b) Visible absorbance difference spectra of cytochrome oxidase: (i) -* * a-, CO-mixed valence compound minus resting; (ii) - *-. - , CO-mixed valence compound minus pulsed. All spectra normalized to cytochrome oxidase concentration 4.6 CM, path length 10 mm.
ferrous heme a3. This reaction is about one hundred times faster than the similar reaction involving the resting enzyme. This appears to be a genuine property of the pulsed enzyme in that the unusual reactivity of the enzyme toward carbon monoxide is lost as the pulsed enzyme returns to its resting state. It seems probable that, as in the case of the resting enzyme, the reductant is the carbon monoxide
l
UTES
f
l l
I
1500
1
loocl
I
500
‘kr--
l
FIGIJRE2. Formationof the CO-mixed valence compound from cytochrome oxidase followed at 431 nm. (Absorbance at t minus absorbance at t = 0, when CO was added.) Note that frames A and B show the same data but with different time scales. (a, b) 0, A, resting cytochrome oxidase; (c) +, pulsed cytochrome oxidase: 45min delay between reoxidation and addition of carbon monoxide;(d) n, pulsed with 220-min delay; (e) 0, pulsed with 360-min delay. All data normalized to cytochrome oxidase concentration 4.6 MM, path length 10 min. Lines are drawn only to guide the eye.
9
0.;
3) 8
w
Pulsed Cytochrome
301
c Oxidase
Oxidized
COamixed
valence
“607nm
complex
*
4202 FIGURE
3.
Carbon monoxide bxygenase cycle proposed by Bickar et al. [13].
itself. The speed of the pulsed enzyme reaction argues against reduction by some endogenous reductant in the sample, as does the fact that the fast reduction goes only as far as the two-electron stage. Since the resting and pulsed forms of the enzyme share the same formal oxidation state, we must look for other factors to account for the divergent rates noted for what otherwise appear to be similar reactions. It has been previously suggested [7] that these two states of the enzyme differ in the ligation of the dioxygen binding site metal centers, and hence in their accessibility to externally added ligands, and possibly also in their redox potentials. Any of these factors could contribute to a difference in the reactivity of the enzyme to carbon monoxide. One intriguing possibility is that the resting enzyme does not react at all, but that there is a slow equilibrium between pulsed and resting forms of the enzyme [20]. The resting enzyme would then be turned into the CO-mixed-valence compound only as it spontaneously became pulsed. This would account for the observation of pulsed-like subpopulations in samples of resting enzyme [7]: very
slow
Resting ,slow
co Pulsed -
CO-Mixed-valence
fast
We have also found that if some oxygen is still present when carbon monoxide is added to the pulsed enzyme, instead of the CO-mixed-valence compound appearing, its oxidation product, the “607-nm complex” is observed first. The “607-nm complex” persists for some time in the visible spectrum and then, presumably when the oxygen is exhausted, quickly gives way to the CO-mixedvalence compound. These observations are explained neatly by a cycle proposed by Bickar et al. [13] (Fig. 3). In this scheme the enzyme is reduced by carbon monoxide to the CO-mixed-valence compound and immediately reoxidized by the oxygen to the “607-nm complex”. Since the CO-mixed-valence compound has two electrons to donate to oxygen, the “607-nm complex” is probably a peroxide adduct at the oxygen binding site. The dissociation or reduction of this peroxide
302
J. E.
Morgan, D. F. Blair and S. I. Chan
adduct could be the slow step in the cycle so that the “607-nm complex” would dominate the spectrum until the oxygen was exhausted, after which the CO-mixedvalence compound would appear. However, Bickar et al. [ 131 proposed this cycle on the basis of the reduction of the resting enzyme by carbon monoxide, a reaction which is far too slow to account for (a) the overall turnover rate of the carbon monoxide oxygenase reaction; (b) the rapid appearance of the CO-mixed-valence compound from the “607-nm complex” when the oxygen runs out [ 131; or (c) the rapid appearance of the “607-nm complex”from the pulsed enzyme in our experiments. The faster reaction involving the pulsed enzyme may explain these observations. In addition, it explains why both Nicholls and Chanady [14] and Young and Caughey [15] found it necessary to prime their enzyme samples with some reductant in order to see the carbon monoxide-oxygenase reactions: the addition of reductant in the presence of oxygen would convert the resting enzyme to pulsed and activate it for fast reduction by carbon monoxide. From the Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena, California 91125. Contribution No. 7124. Supported by grant No. GM22432 from the National Institute of General Medical Sciences, U.S. Public Health Service, and by BRSG grant No. RR7003 awarded by the Biochemical Research Support Grant Program, Division of Research Resources, National Institutes of Health. D. B. was recipient of National Research Service Award 5T32GM-07616 from the National Institute of General Medical Sciences.
REFERENCES 1. 2. 3. 4.
M. Wikstrom, K. Krab, and M. Saraste, Cytochrome Oxidase A Synthesis, Academic Press, London, 1981. J. A. Freedman and S. H. P. Chan, J. Bioenerg. Biomembr. 16, 75 (1984). D. F. Blair, C. T. Martin, J. Gelles, H. Wang, G. W. Brudvig, T. H. Stevens, and S. I. Chan, Chem. Ser. 21, 43 (1983). E. Antonini, M. Brunori, A. Colosimo, C. Greenwood, and M. T. Wilson, Proc. Nat/. Acad. Sci.
USA 74, 3128 (1977). 5. 6.
7. 8. 9. 10. 11.
12. 13. 14. 15. 16.
17. 18. 19. 20.
A. Colosimo, M. Brunori, P. Sarti, E. Antonini, and M. T. Wilson, Israel J. Chem. 21, 30 (1981). J. V. G. Reichardt and Q. H. Gibson, J. Biol. Chem. 257, 9268 (1982). G. W. Brudvig, T. H. Stevens, R. H. Morse, and S. I. Chan, Biochemistry 20, 3912 (1981). F. Armstrong, R. W. Shaw, and H. Beinert, Biochem. Biophys. Acta. 722, 61 (1983). F. G. Fiamingo, R. A. Altschuld, P. P. Moh, and J. 0. Alben, J. Biol. Chem. 257, 1639 (1982). C. Greenwood, M. T. Wilson, and M. Brunori, Biochem. J. 137, 205 (1974). G. M. Clore and E. M. Chance, Biochem. J. 173, 811 (1978). S. Horie, T. Watanabe, and K. Ave. J. Biochem. (Tokyo) 93, 997 (1983). D. Bickar, C. Bonaventura, and J. Bonaventura, J. Biof. Chem. 259, 10777 (1984). P. Nicholls and G. A. Chanady, Biochim. Biophys. Acta, 634, 256 (1981). L. J. Young and W. S. Caughey, in Oxygen and Oxy-Radicals in Chemistry and Biology, M. A. J. Rodgers, and E. L. Powers, Eds., Academic Press, New York, 1981, p. 787. C. R. Hartzell and H. Beinert, Biochim. Biophys. Acta. 368, 318 (1974). D. F. Blair, D. F. Bocian, G. T. Babcock, and S. I. Chan, Biochemistry 21, 6927 (1982). F. G. Halaka, G. T. Babcock, and J. L. Dye, J. Biol. Chem. 257, 1458 (1982). R. M. C. Dawson, D. C. Elliott, W. H. Elliott, and K. M. Jones, Eds., Data for Biochemical Research, Oxford University Press, New York, 1969, pp. 196-197. J. A. Kornblatt and G. H. B. Hoa, Biochemistry 21, 5439 (1982).
Received and accepted November 1984
ABSTRACT When pulsed cytochrome c oxidase is exposed to carbon monoxide in the absence of oxygen the enzyme is converted quickly to its CO-associated mixed valence state. The half-time for this reaction at 0°C is about 4 min. This is about 100 times faster than a similar reaction which begins with the resting form of the enzyme. The possible significance of this reaction in understanding the pulsed/resting phenomenon and the carbon monoxide oxygenase reactions of cytochrome oxidase is discussed.
INTRODUCTION Cytochrome c oxidase is the terminal enzyme in the mitochondrial electron transfer chain; here the respiratory electrons are finally used to reduce dioxygen to water. The enzyme receives four equivalents of electrons from cytochrome c, reduces dioxygen, and conserves the energy from this reaction as a pH gradient across the inner mitochondrial membrane, in part via the uptake of protons in the reduction of oxygen to water, and in part by the electrogenic pumping of protons from the mitochondrial matrix to the cytosol. (For recent reviews on cytochrome oxidase see Refs. l-3.) To accomplish these tasks cytochrome oxidase utilizes four different metal centers: two iron hemes, with the unusual heme A porphyrin, and two coppers. One iron and one copper ion, namely, cytochrome a and copper A, are buried in the protein and do not react with most externally added ligands. These two low potential centers transfer electrons from cytochrome c to the oxygen binding site, and it has been suggested that one of these sites is the primary locus of the proton pumping function. The other two metal centers, cytochrome a3 and copper B, are close together and make up the oxygen binding/reduction site. They are accessible to a number of exogenous ligands in addition to molecular oxygen [3]. Address reprint requests to Dr. Sunney I. Chan, Arthur Amos Noyes Laboratory Institute
of Technology,
Pasadena,
Journal of Inorganic Biochemistry 23, 295-302 (1985) 0 1985 Elsevier Science Publishing Co., Inc. 52 Vanderbilt
of Chemical
Physics,
California
CA 91125.
Ave., New York, NY 10017
295 0162-0134/85/$3.30
296
J. E. Morgan, D. F. Blair and S. I. Chan
Oxidized cytochrome oxidase exhibits at least two states which have different activity. When isolated from the mitochondrion, the enzyme is in a relatively state. Reduction and reoxidation transform the enzyme inactive “resting” temporarily into an activated state known as “pulsed” or “oxygen-pulsed” [4, 51. Both pulsed and resting forms of the oxidized enzyme have the same overall oxidation state, but the pulsed enzyme can be reduced more quickly and reaches steady state turnover sooner than the resting enzyme [5, 61. The pulsed enzyme owes its enhanced activity to faster intramolecular electron transfer between the low potential centers (cytochrome a, copper A) and the oxygen binding site [5]. If the enzyme is not continuously rereduced it decays back to the resting state within a few hours. The pulsed state of the enzyme is also characterized by distinctive features in the visible absorption spectrum which are probably due to cytochrome a3, and which indicate that the oxygen binding site metal centers are in a different ligation state [7, 81. One of the most useful probes in studying the structure of the oxygen binding site is carbon monoxide (CO) [9]. Carbon monoxide binds to reduced cytochrome a3 and stabilizes both cytochrome a3 and copper B in their reduced states. In the carbon monoxide complex, cytochrome a and copper A can either be reduced or oxidized. When both low potential sites are oxidized, the half-reduced state is known as the CO-mixed-valence compound. Since this compound is both photosensitive and reactive with oxygen it has been used as the starting point for a number of kinetic studies 110, 1 I]. The CO-mixed-valence compound has usually been prepared by partial reoxidation of the fully reduced, CO-inhibited enzyme [ 121, but it can be made directly from the resting form of the enzyme by incubating the sample for many hours under carbon monoxide in the absence of oxygen. Evidence has recently been presented [13] that in the latter case the enzyme is being reduced by the carbon monoxide itself in a water-gas shift reaction of the form FeUj3+ +3CO+H,O+ clluz+
Fe,s2+-CO CUB’+
+ CO2 + 2H+
We have found that if carbon monoxide is added to the pulsed rather than to the resting enzyme, the CO-mixed-valence compound is formed in minutes instead of hours. This dramatic difference in the reactivity of the pulsed vs. the resting enzyme towards carbon monoxide is the subject of the present study. We will briefly discuss the manner in which this result might contribute to our understanding of both the pulsed/resting phenomenon and the carbon monoxide oxygenase reactions in which cytochrome oxidase reportedly catalyzes the oxidation of carbon monoxide to carbon dioxide by molecular oxygen [14, 151.
EXPERIMENTAL Beef heart cytochrome c oxidase was prepared by the method of Hartzell and Beinert [16]. Enzyme samples were diluted in a buffer of 50 mM tris and 0.5% Tween-20 (w/v), at pH 7.4. Enzyme concentrations were determined using an extinction coefficient of 39.6/mM/cm at 605 nm 1171. All experiments were performed at ice temperature.
Pulsed Cytochrome
c Oxidase
297
Enzyme samples were made anaerobic by replacing the atmosphere in the cuvette with argon and then agitating to equilibrate the liquid with the atmosphere. Five such cycles were usually used. Argon used for the anaerobic work was made free of oxygen by means of a l-m-long scrubbing column containing manganese dioxide supported on vermiculite. Carbon monoxide (99.99% pure) was obtained from Matheson Co. In preparing the reduced enzyme, NADH was used as the reductant with phenazine methosulfate (PMS) [ 181 as the electron mediator. Both were obtained from Sigma. Reductant solutions were prepared with 1.65 mM NADH and 16.1 PM PMS in 50 mM Hepes buffer pH 7.4. NADH concentrations were determined using a millimolar extinction coefficient of 6220 at 338 nm [19]. The absorbance of the mediator was ignored. Visible absorption spectra were recorded using a Beckman Acta CIII spectrometer interfaced to a Spex Industries SCAMP SC-31 data processor. Spectra were stored on magnetic (floppy) disk. Spectral bandwidth was 1 nm, except for NADH assays when 2 nm was used. Wavelength calibration was carried out with a holomium oxide calibration filter. Preparation of the Pulsed Enzyme Samples of pulsed enzyme were prepared by reducing the enzyme fully (four electrons) and then reoxidizing with oxygen. In preparing the reduced enzyme for this purpose, a great deal of care was taken to use as close as reasonably possible to the exact amount of reductant necessary to reduce the sample fully. If too much reductant is used there may be some left over after reoxidation to rereduce the sample. Since reduced cytochrome oxidase binds carbon monoxide rapidly, this could produce misleading results concerning possible reactions between the pulsed form of the enzyme and carbon monoxide. Too little reductant, on the other hand, would leave some enzyme molecules incompletely reduced and thus produce species other than the pulsed enzyme, containing partly reduced oxygen intermediates. These other species could have very different reactivities towards carbon monoxide from the four-electron pulsed species. In order to achieve precise fourelectron reduction, both the enzyme sample and the reductant solution were first thoroughly deaerated. The reductant solution was then assayed spectrophotometritally and the calculated amount of reductant transferred anaerobically by syringe to the sample cuvette. The enzyme sample was kept at ice temperature until reduction was complete. The reductant assay was necessary because the NADH/ PMS solution is air sensitive and the concentration of reductant may change if all of the air is not removed Our experiments with carbon monoxide required oxygen-pulsed cytochrome oxidase samples which were free of excess oxygen. This was achieved either by adding a very small (close to stoichiometric) amount of oxygen dissolved in water, or by opening the cell to air and then removing the excess oxygen by washing with argon as described above. RESULTS Oxidized cytochrome oxidase is converted into the CO-mixed-valence
compound
298
J. E. Morgan, D. F. Blair and S. I. Chan
when it is incubated under carbon monoxide in the absence of oxygen, This happens whether the enzyme is in its resting or pulsed state (Fig. 1). However, the product appears much more rapidly starting from the pulsed enzyme. Figure 2 shows the time course of these reactions as followed by visible spectroscopy. The half times are about 400 min for the resting enzyme (traces A and B) and about 4 min for the pulsed enzyme (trace C). This unusual reactivity of the pulsed enzyme toward carbon monoxide is lost slowly over the course of several hours. When carbon monoxide is introduced up to 45 min after reoxidation (trace C) there appears to have been little loss of activity, since almost all of the sample is converted quickly to the CO-mixedvalence product. However, if 220 min elapse between reoxidation and addition of carbon monoxide (trace D), the sample behaves like a mixture of pulsed and resting enzyme; that is, the reaction takes place with both a fast phase and a slow phase. If the delay is lengthened to 360 minutes (trace E), the enzyme appears to have returned almost completely to the resting state. It should be noted that there appears to be a small fast component even in the reactions starting with the resting form of the enzyme (traces A and B). The pulsed enzyme used in these experiments was prepared by reducing the resting enzyme with a stoichiometric amount of reductant and then reoxidizing with oxygen. The objective was to produce a large population of the fully oxidized pulsed species in the absence of any excess reductant which might rereduce the enzyme. Avoiding excess reductant is important since reduced cytochrome oxidase binds carbon monoxide very quickly; in the present experiment, if the enzyme was being reduced from some other source, it might still appear that the carbon monoxide was responsible. Control experiments showed that this problem was avoided: When enzyme samples were deliberately reduced with an excess of reductant the resulting pulsed enzyme quickly became fully rereduced (four electrons) in the presence of carbon monoxide; however, when no excess was used, the fast rereduction stopped at the two electron (CO-mixed-valence) stage. It should also be noted that although the samples for the carbon monoxide reaction must be free of oxygen, the pulsed enzyme was produced by aerobic reoxidation; thus, either a very small (close to stoichiometric) amount of oxygen was used, or an excess of oxygen was added and then removed before the addition of carbon monoxide. If some oxygen was still present when carbon monoxide was added to the pulsed enzyme, the CO-mixed-valence compound was not seen immediately. Instead, the spectrum of a species known as the “607-nm complex” [14] was observed. The “607-nm complex” is the species produced when the CO-mixed-valence compound reacts with dioxygen and is so named because it has a peak at 607 nm when compared to the resting enzyme. The “607-nm complex” persisted for some time in the visible spectrum and then, presumably when the oxygen was exhausted, the CO-mixed-valence compound appeared quickly.
DISCUSSION We have shown that in the absence of oxygen, carbon monoxide rapidly converts pulsed cytochrome oxidase into the CO-mixed-valence compound, a process which involves both reduction by two electrons and binding of carbon monoxide to
Msed
Cytochrome c Oxidase
700
299
600
500 WAVELENGTH
4co
J
(nm)
FIGURE 1.
(a) Visible absorbance spectra of cytochrome ox&se: (i) . . . , resting; (ii) -, pulsed; (iii) - - -, Co-mixed valence compound. All spectra normalized to cytochrome oxidase concentration 4.6 CM, path length 10 mm. (b) Visible absorbance difference spectra of cytochrome oxidase: (i) -* * a-, CO-mixed valence compound minus resting; (ii) - *-. - , CO-mixed valence compound minus pulsed. All spectra normalized to cytochrome oxidase concentration 4.6 CM, path length 10 mm.
ferrous heme a3. This reaction is about one hundred times faster than the similar reaction involving the resting enzyme. This appears to be a genuine property of the pulsed enzyme in that the unusual reactivity of the enzyme toward carbon monoxide is lost as the pulsed enzyme returns to its resting state. It seems probable that, as in the case of the resting enzyme, the reductant is the carbon monoxide
l
UTES
f
l l
I
1500
1
loocl
I
500
‘kr--
l
FIGIJRE2. Formationof the CO-mixed valence compound from cytochrome oxidase followed at 431 nm. (Absorbance at t minus absorbance at t = 0, when CO was added.) Note that frames A and B show the same data but with different time scales. (a, b) 0, A, resting cytochrome oxidase; (c) +, pulsed cytochrome oxidase: 45min delay between reoxidation and addition of carbon monoxide;(d) n, pulsed with 220-min delay; (e) 0, pulsed with 360-min delay. All data normalized to cytochrome oxidase concentration 4.6 MM, path length 10 min. Lines are drawn only to guide the eye.
9
0.;
3) 8
w
Pulsed Cytochrome
301
c Oxidase
Oxidized
COamixed
valence
“607nm
complex
*
4202 FIGURE
3.
Carbon monoxide bxygenase cycle proposed by Bickar et al. [13].
itself. The speed of the pulsed enzyme reaction argues against reduction by some endogenous reductant in the sample, as does the fact that the fast reduction goes only as far as the two-electron stage. Since the resting and pulsed forms of the enzyme share the same formal oxidation state, we must look for other factors to account for the divergent rates noted for what otherwise appear to be similar reactions. It has been previously suggested [7] that these two states of the enzyme differ in the ligation of the dioxygen binding site metal centers, and hence in their accessibility to externally added ligands, and possibly also in their redox potentials. Any of these factors could contribute to a difference in the reactivity of the enzyme to carbon monoxide. One intriguing possibility is that the resting enzyme does not react at all, but that there is a slow equilibrium between pulsed and resting forms of the enzyme [20]. The resting enzyme would then be turned into the CO-mixed-valence compound only as it spontaneously became pulsed. This would account for the observation of pulsed-like subpopulations in samples of resting enzyme [7]: very
slow
Resting ,slow
co Pulsed -
CO-Mixed-valence
fast
We have also found that if some oxygen is still present when carbon monoxide is added to the pulsed enzyme, instead of the CO-mixed-valence compound appearing, its oxidation product, the “607-nm complex” is observed first. The “607-nm complex” persists for some time in the visible spectrum and then, presumably when the oxygen is exhausted, quickly gives way to the CO-mixedvalence compound. These observations are explained neatly by a cycle proposed by Bickar et al. [13] (Fig. 3). In this scheme the enzyme is reduced by carbon monoxide to the CO-mixed-valence compound and immediately reoxidized by the oxygen to the “607-nm complex”. Since the CO-mixed-valence compound has two electrons to donate to oxygen, the “607-nm complex” is probably a peroxide adduct at the oxygen binding site. The dissociation or reduction of this peroxide
302
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adduct could be the slow step in the cycle so that the “607-nm complex” would dominate the spectrum until the oxygen was exhausted, after which the CO-mixedvalence compound would appear. However, Bickar et al. [ 131 proposed this cycle on the basis of the reduction of the resting enzyme by carbon monoxide, a reaction which is far too slow to account for (a) the overall turnover rate of the carbon monoxide oxygenase reaction; (b) the rapid appearance of the CO-mixed-valence compound from the “607-nm complex” when the oxygen runs out [ 131; or (c) the rapid appearance of the “607-nm complex”from the pulsed enzyme in our experiments. The faster reaction involving the pulsed enzyme may explain these observations. In addition, it explains why both Nicholls and Chanady [14] and Young and Caughey [15] found it necessary to prime their enzyme samples with some reductant in order to see the carbon monoxide-oxygenase reactions: the addition of reductant in the presence of oxygen would convert the resting enzyme to pulsed and activate it for fast reduction by carbon monoxide. From the Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena, California 91125. Contribution No. 7124. Supported by grant No. GM22432 from the National Institute of General Medical Sciences, U.S. Public Health Service, and by BRSG grant No. RR7003 awarded by the Biochemical Research Support Grant Program, Division of Research Resources, National Institutes of Health. D. B. was recipient of National Research Service Award 5T32GM-07616 from the National Institute of General Medical Sciences.
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Received and accepted November 1984