The Heme Groups of Cytochrome O from Escherichia Coli
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Proc. Natl. Acad. Sci. USA Vol. 88, pp. 6122-6126, July 1991 Biochemistry The heme groups of cytochrome o from Escherichia coli (cytochrome oxidase/quinol oxidase/heme A/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 ubiquinol 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 enzyme 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 prosthetic group of b-type cytochromes, oxidized a band, including its 555- and 561-nm components, including cytochrome o. However, the hemes 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 redox 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).