Proc. Natl. Acad. Sci. USA Vol. 88, pp. 6122-6126, July 1991 Biochemistry The groups of o from (cytochrome oxidase/quinol oxidase//heme 0) ANNE PUUSTINEN AND MARTEN WIKSTROM Helsinki Bioenergetics Group, Department of Medical Chemistry, University of Helsinki, Siltavuorenpenger 10A, SF-00170 Helsinki, Finland Communicated by Britton Chance, April 4, 1991 (receivedfor review January 16, 1991)

ABSTRACT Cytochrome o, one of the two terminal tential (265 mV). The other heme species exhibited an Em of oxidases of Escherichia cofi, is structurally and 140 mV. On the basis of the potentiometric behavior of the functionally related to cytochrome c oxidase of mitochondria high-spin signal of the oxygen-reacting heme, Salerno et al. and some bacteria. It has two heme groups, one ofwhich binds (7, 12) concluded that the Em for Cu in the binuclear site is CO and forms a binuclear oxygen reaction center with copper. =350 mV. They further demonstrated that the ex- The other heme is unreactive toward ligands, exhibits strong hibits anticooperative heme-heme and high-spin heme- interactions with the binuclear center, and is mainly respon- copper interactions, similar to those in cytochrome aa3. sible for the reduced-minus-oxidized a band. Protoheme has We recently suggested that the entire reduced-minus- been thought to be the of b-type , oxidized a band, including its 555- and 561-nm components, including cytochrome o. However, the of cytochrome o is due almost entirely to the six-coordinated low-spin heme, are of a different kind, for which we propose the name heme whereas both hemes contribute about equally to the Soret 0. Its pyridine hemochrome spectrum is blue-shifted by 4 nm band (6). However, the a band ofthe reduced-minus-oxidized relative to that of protoheme, and chromatographic behavior enzyme behaves inhomogeneously, both spectrally and po- showed that it is much more hydrophobic than protoheme. Fast tentiometrically (12, 13). With Salerno et al. (7, 12), we atom bombardment mass spectrometry yielded a molecular ascribe this to combined spectral and potential inter- mass of 839 Da. Heme 0 is proposed to be a heme A-like actions between the low-spin heme and the binuclear center. molecule, containing a 17-carbon hydroxyethylfarnesyl side CO has been described to have small and somewhat chain, but with a methyl residue replacing the formyl group. variable effects on the spectrum ofthe reduced enzyme in the a band (11, 14-17). Here we study the CO-difference spec- Escherichia coli contains two terminal oxidases, both of trum in some detail and report its relevant specific absorp- which oxidize ubiquinol by molecular oxygen. One of them tivities. was called cytochrome o (for oxidase) by Castor and Chance The hemes ofcytochrome o have been generally thought to (1), who described its CO-binding properties. The genes for be protohemes (see e.g., refs. 18 and 19). However, scruti- cytochrome o have been elucidated (2), and strong protein- nization of the cited data indicates that an actual determina- structural homology has been found with cytochrome c tion ofthe heme type has not been made previously* and that oxidase (cytochrome aa3) of mitochondria and some bacteria the anomalously blue-shifted pyridine hemochrome (6) has (2, 3), especially for the heme-binding largest subunit. The been unnoticed or neglected. Here we propose a structure for other quinol oxidase of E. coli, cytochrome d (or bd), is the prosthetic heme groups of cytochrome o. structurally and functionally unrelated to cytochrome aa3 (4-6). Cytochrome o contains two hemes. One binds CO (1) MATERIALS AND METHODS and probably forms a binuclear 02 reaction center together with a copper ion (7). The other heme is probably a low-spin Deoxycholate-washed (20) membranes from E. coli strain RG six-coordinated hemochrome, as revealed both by its EPR 145 (cyd-) (ref. 21; for growth conditions see ref. 6) were and optical spectra (6-8). Cytochrome o has only one copper further purified with cold acetone/NH40H, and the hemes per two hemes and lacks the CUA center typical of cy- were extracted from the pellet into cold acetone/HCI as tochrome aa3 (6, 9). It functions as a proton pump much like described by Weinstein and Beale (22). Alternatively, prep- cytochrome aa3 (6, 10). arations ofpurified cytochrome o (6), sperm whale myoglobin Thus cytochrome o is both structurally and functionally (Sigma), or bovine heart cytochrome oxidase (23) were used. homologous to cytochrome aa3, but it exhibits distinct dif- The hemes in acetone/HCI were extracted into ether, and the ferences as well. We have undertaken studies ofcytochrome heme-containing upper phase was washed with water (22). o to gain more insight into the structure and function of The ether was evaporated under a stream of nitrogen. The terminal proton-translocating oxidases, by their comparison. hemes were dissolved in ethanol/methylene chloride (50:50, In this paper we report on the spectral, redox, and structural vol/vol) and applied to a 1.5-ml bed volume column of properties of the heme groups. DEAE-Sepharose CL-6B (acetate) (24), which had been The spectral properties of cytochrome o have been con- equilibrated with ethanol/methylene chloride (25). The col- troversial. The reduced-minus-oxidized a band is split at 77 umn was washed with 20 ml of the equilibration solution, K into peaks at -555 and 561 nm. The 555-nm species has followed by 3 ml of aqueous ethanol, and the hemes were been ascribed to the CO-reactive high-spin heme (11, 12), eluted in ethanol/acetic acid/water (70:17:13, vol/vol) (25). which according to Salerno et al. (12) has a midpoint redox The hemes were separated from each other by reverse- potential relative to the normal hydrogen electrode (Em) at phase HPLC by using an Altex Ultrasphere ODS column (25 pH 7 of -160 mV. On the other hand, Withers and Bragg (13) cm x 4.6 mm) with 5-,um particles. The solvent was 95% attributed the long wavelength species to the oxygen-reactive heme, to which they ascribed a CO-sensitive midpoint po- Abbreviations: Em, midpoint redox potential relative to the normal hydrogen electrode; TMPD, tetramethyl-p-phenylenediamine. *In the course of this work we noted that Castor and Chance (1), The publication costs of this article were defrayed in part by page charge referring to unpublished work of L. Smith, reported that the hemes payment. This article must therefore be hereby marked "advertisement" of "cytochrome o" are not protohemes. To our knowledge this has in accordance with 18 U.S.C. §1734 solely to indicate this fact. been overlooked and not followed up subsequently. 6122 Downloaded by guest on September 29, 2021 Biochemistry: Puustinen and Wikstr6m Proc. Natl. Acad. Sci. USA 88 (1991) 6123 A ethanol/acetic acid/water (70:17:7, vol/vol), and the flow rate was 0.6 ml/min. Heme fractions were detected by absorbance at 402 nm, collected, and evaporated in a rotary evaporator. ll+ Fast atom bombardment mass spectra were recorded at the AA = AA= State Technical Research Center (VTT; Espoo, Finland) by 0.02 0.002 using a JEOL SX 102 mass spectrometer. HPLC-purified f protoheme (from myoglobin) and heme 0 (see above) were dissolved into a glycerol matrix on the sample plate. The sample was bombarded by Xe atoms at an acceleration voltage of 10 kV and a fast atom bombardment gun voltage of 3 kV, using the positive ion mode. The temperature of the ion source was 530C, and the scan range was 20-2000 m/z. The data was processed with a Hewlett-Packard 9000 com- . ,puter. Optical spectra were recorded using a Shimadzu UV-3000 instrument. Dual wavelength spectrophotometry was carried Il Il out by using a DBS-1 (Johnson Foundation Workshop, University of Pennsylvania) spectrophotometer. All optical I ...... spectroscopy was performed at room temperature in cuvettes .1 with a 1-cm light path length. CO-difference spectra were obtained by first recording the baseline ofthe anaerobically reduced sample in the Shimadzu (Kyoto) instrument at a 0.5-nm slit width [tetramethyl-p- 400 440 480 520 560 600 phenylene diamine (TMPD) plus ascorbate, see the legend to Wavelength, nm Fig. 1], by subsequent slow bubbling of CO gas through the sample for 6 min in the dark, and by finally recording the B difference spectrum. A second spectrum, routinely recorded after a further CO treatment for 6 min, showed no difference T from the first. AA = Pyridine hemochrome spectra were recorded as described 0.002 by Berry and Trumpower (26). A 1 AI RESULTS AND DISCUSSION of the Low- and Hemes of T 1U>, Spectral Properties High-Spin AA = " __ Cytochrome o. Fig. 1A shows the reduced-minus-oxidized TAA_ difference spectrum ofthe purified cytochrome o preparation 0.01 \AA = I (6). It is important to note that the enzyme was reduced 0.001 anaerobically with TMPD plus ascorbate (see below). The a band is broad at room temperature; it is known from previous work that two peaks can be distinguished at 77 K (11, 12, 17). The Soret/a-band ratio is 11.8, in the same range as found by , others (10.6-12.2; cf. refs. 1, 11, and 15), but much higher 400 440 480 520 56050 600k than for cytochrome aa3 (27). The specific absorptivity ofthe Wavelength, nm a band (reduced minus oxidized; 560 minus 580 nm) is about 24 mM-1 cm-1 on the basis of concentration of enzyme (all C specific absorptivities reported here are based on the con- centration of enzyme, containing two hemes), which was in AA = 0.001 turn deduced from pyridine hemochrome determination (6). 556-565 nm The corresponding value for the Soret band (reduced minus f oxidized; 427 minus 460 nm) is then 283 mM-1 cm-1. This is similar to values calculated from spectra reported in refs. 11 [02N 0 and 15. The CO-difference spectrum (Fig. 1A) of the reduced 1 min P., cytochrome o is very similar to the photochemical action spectrum measured by Castor and Chance (1). It is important FIG. 1. Reduced-minus-oxidized and CO-difference spectra of to note that the red region ofthe spectrum does not reveal any isolated cytochrome o. The medium contained 40 nnM Tris Cl, 0.05% distinct trough below the baseline, but it shows discrete a and (wt/vol) dodecyl maltoside at pH 7.4, and 0.44 ,uM cytochrome o. (A) P3 bands at 568 and 535 nm, respectively. This supports the The baseline was recorded .... ), followed by aaddition of 2 mM previous contention that the CO-reactive high-spin heme ascorbate and 0.4 mM TMPD. After anaerobiosis, the reduced- contributes little, if any, to the reduced-minus-oxidized dif- minus-oxidized difference spectrum was recordedi- . nen tnis spectrum was memorized as a new baseline, akd the sample was treated with CO (see Materials and Methods), an(d the CO-reduced recorded ( ). (C) The time course of the aerobic-anaerobic minus reduced spectrum was recorded (----). (I 9) The conditions transition was followed at 556-565 nm. The medium was as described were identical to those in A, except for the concent.ration of enzyme, above; TMPD, ascorbate, and enzyme concentrations were 0.4 mM, which was 0.33 ,uM. The anaerobically reduced (minus oxidized) 10 mM, and 0.8 uM, respectively. Points of anaerobiosis are indi- spectrum was recorded as before (----) and mem(orized. After this, cated; at +02, dioxygen was added by stirring. The absorbance a few grains ofsolid sodium dithionite were added, aand the dithionite- increases at 556 and 565 nm correspond to a downward and upward reduced minus anaerobically reduced difference spectrum was deflection, respectively. Downloaded by guest on September 29, 2021 6124 Biochemistry: Puustinen and Wikstrom Proc. Natl. Acad Sci. USA 88 (1991) ference spectrum in the 500- to 600-nm region (6). Its con- whether the heme (and/or copper) of the binuclear site is tribution to the Soret band is near 50% (Fig. PA). oxidized or reduced. In the former case, the Em is high and The a and (3 peaks of the CO-difference spectrum both the a-absorption peak(s) are red-shifted; in the latter case, the have specific absorptivities of about one-half ofthe reduced- opposite is true. The splitting of the 560-nm band at 77 K is minus-oxidized 560-nm band. Here we differ from both Kita then probably due to an asymmetrical optical transition, also et al. (11) and Matsushita et al. (15), who found substantially exhibited by the low-potential b-type heme ofthe cytochrome lower specific absorptivities. We find the peak-to-trough bc, complex (30). absorptivity ofthe Soret CO-difference spectrum to be about Cytochrome o Contains Heme 0. The hemes of cytochrome 287 mM-1cm-1. This is again much higher than values o have been reported previously (11, 15, 28). However, the ratio of the thought to be protohemes as is thought to peak-to-trough in the Soret band to the extent of the a peak generally be the case for the hemes of b-type cytochromes is 23.7 (Fig. P1), which is similar to earlier data (11, 15, 28). (see e.g., ref. 31). Hemes extracted from cytochrome o and Therefore, we ascribe the higher molar absorptivities found from membranes ofE. coli (see Materials andMethods) were here to more complete occupancy of the CO adduct. run on an HPLC column that readily separates protoheme A Contaminating, Low-Potential -556. In con- from heme A. As seen in Fig. 2, our enzyme preparation trast to the experiment above, reduction of cytochrome o for contains two kinds of heme, one of which runs identically spectroscopic analysis has usually been routinely performed with protoheme, but it accounts for only -25-30%o of the with dithionite. Addition of dithionite to the reduced anaer- total. The main heme fraction (heme 0, see below) is highly obic sample caused further reduction of a cytochrome b hydrophobic; it is retained on the column even longer than component with a sharp a-peak maximum at 556 nm and a heme A. The amount of protoheme coincides with the Soret maximum at 424 nm in the difference spectrum (Fig. optically determined amount of low-potential cytochrome 1B). This species is also hardly reduced anaerobically with b-556 species described above. In preliminary work, cy- ubiquinol 1 and does not react with CO. Apparently it has an tochrome b-556 has been separated from the cytochrome o Em significantly lower than that of the other species. Its entity; pyridine hemochrome determinations have confirmed absorbance varies between 20o and 30%o of the total dithio- that b-556 indeed contains protoheme (A.P., unpublished nite-reduced minus oxidized a band in our enzyme prepara- results). tions. It is thus clearly substoichiometric with respect to the cytochrome o hemes (see also below). The cytochrome b-556 has probably contributed to spectral and potentiometric multiplicity of b cytochromes in at least some cytochrome o A preparations reported previously and to variations in such multiplicity between different enzyme preparations (see e.g., ref. 13). We ascribe cytochrome b-556 to a low-potential b-type cytochrome that may contaminate most cytochrome o preparations. It might yet be of functional importance for quinol oxidase activity (see below). Kita et al. (29) have purified and characterized a b-type cytochrome (which they called b-556) from E. coli, which has spectral and redox properties very similar to those of cytochrome b-556 de- scribed here. The Basis for the Asymmetric Behavior of the 560-nm Band. Fig. 1C shows that, upon anaerobiosis with TMPD plus ascorbate, the 560-nm band (Fig. 1 A and B) is formed asymmetrically. This was determined by the dual-wavelength technique with the two wavelengths symmetrically displaced from the center of the absorption band. The absorbance increase at anaerobiosis starts at the longer wavelengths of this band (initial upward deflection at 02 = 0), followed by a B slower (later) absorbance increase at the shorter wavelengths (secondary downward deflection). These kinetics do not involve the b-556 component, which is insignificantly re- duced in these conditions and on this time scale. This result suggests a spectral contribution to the 560-nm band from at least two heme components: a long-wavelength species with high Em and a short-wavelength species with lower Em. Either both hemes of the enzyme contribute significantly to the 560-nm band, or then there must be a combination of spectral and redox interactions between the low-spin heme and the binuclear oxygen reaction site. Since we have ex- cluded the former possibility (above), the latter must be the case. -I Previously, the 02- and CO-binding heme has been vari- 10 20 30 40 50 60 70 80 ably assigned to the long- and short-wavelength components Time, min of the 560-nm band, respectively (see Introduction), based on the and heterogeneous behav- FIG. 2. Reverse-phase HPLC elution profiles of hemes. The spectrally potentiometrically hemes were extracted from purified cytochrome o (A) and from ior of the band, also reported here, its splitting at 77 K, and myoglobin and cytochrome c oxidase, the hemes of which were the CO-difference spectrum. On the basis of our data, we combined (B). Elution times for protoheme, heme A, and heme 0 ascribe the heterogeneity exclusively to center-center inter- were 29, 61, and 71 min, respectively. In A, the integrated areas of actions in the enzyme (see also ref. 7). Thus both the Em and the protoheme and heme 0 peaks were 27% and 63%, respectively, the position of the a band of the low-spin heme depend on of the total integrated heme absorbance at 402 nm. Downloaded by guest on September 29, 2021 Biochemistry: Puustinen and Wikstr6m Proc. Natl. Acad. Sci. USA 88 (1991) 6125

fI hematoheme, where positions 2 and 4 are both occupied by I I hydroxyethyl groups (34). This comparison suggests that one I I but not both vinyl groups of protoheme has been replaced in I I a I I heme 0 by a less electron-attracting residue, possibly I I (substituted) hydroxyethyl group. I I Protoheme is one of the most hydrophobic naturally oc- I curring hemes. In this respect, it is surpassed only by heme I A, the hydrophobicity of which is mainly due to its long I hydroxyalkyl side chain. The behavior of heme 0 on HPLC I (Fig. 2) therefore strongly suggests the presence of a similar I side chain. Combining this with the information from the I T pyridine hemochrome spectrum allows the conjecture that I A = 0.005 heme 0 might differ from protoheme by having one vinyl I group ofthe latter replaced by a long hydroxyalkyl side chain. I A minimum hypothesis would further suggest that heme 0 I may have a methyl group in position 8, as in protoheme, in I place of the formyl group of heme A. I I A long hydroxyalkyl side chain would increase the molec- I ular mass of heme 0 much above that of protoheme. Fast atom bombardment mass spectrometry of isolated and HPLC-purified protoheme from myoglobin yielded a mass of 617 Da for the molecular ion, which is the expected value. The corresponding mass of purified heme 0 was 839 Da, which strongly suggests the presence of a long alkyl side chain. The molecular mass of heme 0 is 14 Da smaller than that of heme A. This is consistent with a heme 0 structure that differs from heme A only by having the formyl group of the latter replaced by a methyl group. Thus heme 0 is proposed to have a hydroxyethylfamesyl side chain identical to that in heme A in one of the positions (2 or 4) occupied by 510 530 550 570 590 a vinyl group in protoheme. The analogy to heme A makes Wavelength, nm substitution at position 2 more likely. A methyl group in position 8, instead of formyl, could explain why heme 0 FIG. 3. Dithionite-reduced minus oxidized pyridine hemochrome appears more hydrophobic than heme A. We conclude that spectra of heme 0 ( ) and protoheme (----) from HPLC-purified the proposed structure is consistent with all the available heme fractions. data, but that it still needs to be proven unambiguously by NMR spectroscopy. Fig. 3 shows that the pyridine hemochrome spectrum of Conclusions. We conclude that the cytochrome o entity purified heme 0 is blue-shifted by 4 nm relative to proto- contains two hemes of the 0 type. Our enzyme preparation heme. The hemes of cytochrome o must have a structure further contains variable, substoichiometric amounts of a different from protoheme, which we tentatively call heme 0.t low-potential cytochrome b-556 species, the prosthetic group A Possible Structure of Heme 0. Pyridine hemochrome of which is protoheme. This species is also present in at least spectra are very sensitive to the nature and number of some ofthe cytochrome o preparations described earlier (13). electron-attracting groups on the porphyrin ring, and this We have found that the b-556 can be separated from the phenomenon has been extensively studied in the past (see ref. cytochrome o entity; this appears to be associated with a 34). For example, we can readily exclude the presence of a decrease of ubiquinol oxidase activity (A.P., unpublished formyl group, which due to its very strong electron-attracting results). Cytochrome b-556 might be the entity that oxidizes power displaces pyridine hemochrome and native spectra far quinol and delivers the electrons to the cytochrome o com- to the red, as in heme A. The pyridine hemochrome spectrum plex. of protoheme is much less red-shifted, but the peak position The two heme 0 groups have different properties when is yet further to the red than for many other hemes. This is bound to the enzyme (cf. hemes A in cytochrome aa3). We due to the electron-attracting vinyl groups in the 2 and 4 conclude that the low-spin heme is the main contributor to the positions of the porphyrin ring (34). The pyridine hemo- a band of reduced-minus-oxidized enzyme with very little, if chrome spectrum of heme 0 is blue-shifted by 4 nm from this any, contribution from the high-spin CO- and 02-binding (Fig. 3) and is very similar to that of 2(4)-hydroxyethyl-4(2)- heme. In the Soret, both hemes contribute nearly equally to vinyl deuteroheme. However, the blue shift is smaller than in the reduced-minus-oxidized band. Yet, the a band behaves heterogeneously both spectrally and as a redox system. We tAccording to current convention, both isolated heme structures and ascribe this to interactions between the low-spin heme and cytochromes are named a, b, c, and d (31). However, in agreement the binuclear heme-copper center (cf. refs. 7 and 12). All with Caughey et al. (32), we prefer the use of capital letters to these properties are also typical for the cytochrome aa3 describe the heme structure as isolated (33). Lowercase letters may system (33). then be freely used for cytochromes and , as well as to The discovery of heme 0 in cytochrome o provides yet describe individual protein-bound heme groups (for example, cy- These tochrome bc, and aa3 complexes, cytochrome b5, heme cl of the bc, another similarity to cytochrome aa3-type oxidases. complex, heme a3 of the aa3 complex, etc). On the basis of the now comprise primary protein structure, especially subunit I strong homology between cytochrome o and cytochrome aa3-type (2, 3), function (proton pumping; ref. 10), and the 02 reduc- oxidases and the distinct heme 0 structure of the former, we tion site (7). Major differences are the electron donor and the tentatively suggest that cytochrome o be termed cytochrome 003, absence of the CUA center from the ubiquinol oxidase (2, 3, which also appears historically pertinent. Thus heme o is the low-spin hemochrome-type species that does not react with ligands 6, 9). As suggested here, the only difference in heme structure and heme 03 is the high-spin CO- and 02-reactive heme of the may be the replacement of the formyl group of heme A by a binuclear site, both having the heme 0 structure as isolated. methyl group in heme 0. This is expected to lower the Em Downloaded by guest on September 29, 2021 6126 Biochemistry: Puustinen and Wikstrom Proc. Natl. Acad. Sci. USA 88 (1991) value, which is borne out experimentally (7). Interestingly, Conference Short Reports (Elsevier, Amsterdam), Vol. 6, p. 22. the well-conserved tyrosine residue of aa3-type oxidases, 10. Puustinen, A., Finel, M., Virkki, M. & Wikstrom, M. (1989) which has been implicated in forming a hydrogen bond to the FEBS Lett. 249, 163-167. 11. Kita, K., Konishi, K. & Anraku, Y. (1984) J. Biol. Chem. 259, formyl of heme a (35), is replaced by leucine in cytochrome 3368-3374. o (2). In bacteria (e.g., Paracoccus denitrificans) that express 12. Salerno, J. C., Bolgiano, B. & Ingledew, W. J. (1989) FEBS cytochromes o and aa3 simultaneously, this difference may Lett. 247, 101-105. be essential for insertion of the correct prosthetic group. 13. Withers, H. K. & Bragg, P. D. (1990) Biochem. Cell Biol. 68, Paracoccus denitrificans membranes indeed contain heme 83-90. on 14. Poole, R. K. (1983) Biochim. Biophys. Acta 726, 205-243. 0, based HPLC analysis (A.P., unpublished results), 15. Matsushita, K., Patel, L. & Kaback, H. R. (1984) Biochemistry suggesting that this heme is not unique for cytochrome o from 23, 4703-4714. E. coli. 16. Yang, T. Y. & Jurtshuk, P., Jr. (1978) Biochem. Biophys. Res. The function of the long hydroxyethylfarnesyl side chain Commun. 81, 1032-1039. might simply be to anchor the prosthetic groups tightly to the 17. Poole, R. K. & Ingledew, W. J. (1987) in E. coli and S. membranous enzyme, without covalent bonding. But it is not typhimurium: Cellular and Molecular Biology, ed. Neidhardt, understood why this is unnecessary in the cytochrome bc1 F. C. (Am. Soc. Microbiol., Washington), Vol. 1, pp. 170-200. 18. Nakamura, H., Yamato, I., Anraku, Y., Lemieux, L. & Gen- complex, for example, where the b-type cytochromes contain nis, R. B. (1990) J. Biol. Chem. 265, 11193-11197. protoheme (A.P., unpublished results). However, it might be 19. Minagawa, J., Nakamura, H., Yamato, I., Mogi, T. & Anraku, related to the more complicated chemistry catalyzed by the Y. (1990) J. Biol. Chem. 265, 11198-11203. oxidases, with respect to both 02 reduction and proton- 20. Ludwig, B. (1986) Methods Enzymol. 126, 153-159. pumping. These functions might require structural transitions 21. Au, D. C.-T. & Gennis, R. B. (1987) J. Bacteriol. 169, 3237- during the catalytic cycle that necessitate tight anchoring of 3242. the heme groups. 22. Weinstein, J. D. & Beale, S. I. (1983) J. Biol. Chem. 258, 6799-6807. 23. Yu, C., Yu, L. & King, T. E. (1975) J. Biol. Chem. 250, We thank Hilkka Vuorenmaa for help in preparation of the 1383-1392. manuscript and Pentti Somerhaiju for putting his HPLC system at 24. Omata, T. & Murata, N. (1980) Photochem. Photobiol. 31, our disposal. This work was supported by grants from the Sigrid 183-185. Juselius Foundation and the Academy of Finland. 25. Weinstein, J. D., Branchaud, R., Beale, S. I., Bement, W. J. & Sinclair, P. R. (1986) Arch. Biochem. Biophys. 245, 44-50. 1. Castor, L. N. & Chance, B. (1959) J. Biol. Chem. 234, 1587- 26. Berry, E. A. & Trumpower, B. L. (1987) Anal. Biochem. 161, 1592. 1-15. 2. Chepuri, V., Lemieux, L., Au, D. C.-T. & Gennis, R. B. (1990) 27. Vanneste, W. H. (1966) Biochemistry 5, 838-848. J. Biol. Chem. 265, 11185-11192. 28. Daniel, R. M. (1970) Biochim. Biophys. Acta 216, 328-341. 3. Saraste, M., Raitio, M., Jalli, T., Chepuri, V., Lemieux, L. & 29. Kita, K., Yamato, I. & Anraku, Y. (1978) J. Biol. Chem. 253, Gennis, R. B. (1988) Ann. N. Y. Acad. Sci. 550, 314-324. 8910-8915. 4. Kita, K., Konishi, K. & Anraku, Y. (1984) J. Biol. Chem. 259, 30. Sato, N., Wilson, D. F. & Chance, B. (1971) Biochim. Biophys. 3375-3381. Acta 253, 88-97. 5. Anraku, Y. & Gennis, R. B. (1987) Trends Biochem. Sci. 12, 31. International Union of Biochemistry (1979) Enzyme Nomen- 262-266. clature (Academic, New York), pp. 593-601. 6. Puustinen, A., Finel, M., Haltia, T., Gennis, R. B. & Wik- 32. Caughey, W. S., Smythe, G. A., O'Keefe, D. H., Maskasky, str6m, M. (1991) Biochemistry 30, 3936-3942. J. E. & Smith, M. L. (1975) J. Biol. Chem. 250, 7602-7622. 7. Salerno, J. C., Bolgiano, B., Poole, R. K., Gennis, R. B. & 33. Wikstrom, M., Krab, K. & Saraste, M. (1981) Cytochrome Ingledew, W. J. (1990) J. Biol. Chem. 265, 4364-4368. Oxidase-A Synthesis (Academic, New York). 8. Hata, A., Kirino, Y., Matsuura, K., Itoh, S., Hiyama, T., 34. Falk, J. E. (1964) Porphyrins and Metalloporphyrins (Elsevier, Konishi, K., Kita, K. & Anraku, Y. (1985) Biochim. Biophys. Amsterdam). Acta 810, 62-72. 35. Holm, L., Saraste, M. & Wikstrom, M. (1987) EMBO J. 6, 9. Puustinen, A. & Wikstrom, M. (1990) European Bioenergetics 2819-2823. Downloaded by guest on September 29, 2021