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Copper Electron-Nuclear Double Resonance of Cytochrome C Oxidase (Electron Paramagnetic Resonance) BRIAN M

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Copper Electron-Nuclear Double Resonance of Cytochrome C Oxidase (Electron Paramagnetic Resonance) BRIAN M Proc. Nati. Acad. Sci. USA Vol. 77, No. 3, pp. 1452-1456, March 1980 Biophysics Copper electron-nuclear double resonance of cytochrome c oxidase (electron paramagnetic resonance) BRIAN M. HOFFMAN*t, JAMES E. ROBERTS*, MAURICE SWANSONt, SAMUEL H. SPECKt, AND E. MARGOLIASHt Departments of *Chemistry and tBiochemistry and Molecular Biology, Northwestern University, Evanston, Illinois 60201 Contributed by Emanuel Margoliash, December 5,1979 ABSTRACT Electron-nuclear double resonance of copper EXPERIMENTAL PROCEDURES was observed while monitoring the "intrinsic copper" electron paramagnetic resonance signal of cytochrome c oxidase (fer- Cytochrome c oxidase was prepared from beef heart mito- rocytochrome c:oxygen. oxi oreductase, EC 1.9.3.1) near g = 2. chondria according to Hartzell and Beinert (14) and was stored This unambiguously establishes the presence of the metal (Cu.) in liquid nitrogen in 0.02 M Tris.HCI, pH 7.4/0.25% Tween 20. in the 'redox center responsible for this signal. The hyperfine It had an enzymic turnover of 350 electrons per sec as deter- couplings to copper are largely isotropic and the maximum value is about half that seen in type I blue copper proteins. The mag- mined by the method of Nicholls et al. (15). Heme a concen- netic properties of this oxidized copper center are not consistent tration was determined spectrophotometrically by using a with those of a thiyl radical (R-S.) coordinated to Cu(I), and Ac605-6,0 = 13.1 mM-1 cm-1 (16). All EPR and ENDOR thus favor the identification of this redox center as a Cu(II) ion spectra were recorded on enzyme preparations that were di- in a unique environment. luted by 50% with glycerol and contained a final concentration Cytochrome c oxidase (ferrocytochrome c:oxygen oxidore- of 0.25 mM cytochrome aa3. ductase, EC 1.9.3.1), the terminal enzyme complex of the eu- ENDOR experiments were performed at 2.0K, using the karyotic electron transport chain, is a transmembrane oli- spectrometer described elsewhere (13), to which has been added gomeric protein consisting of six to ten polypeptides containing a WAVETEK model 2001 sweep generator and a Fabritek two a-type hemes and two copper atoms, as well as lipid (1, 2). model 1074 signal averager. ENDOR spectra were obtained Electron paramagnetic resonance (EPR) spectra of cytochrome with a 100-kHz filed modulation amplitude of -4 G (1 G = oxidase exhibit signals from several paramagnetic centers (3-5). 10-4 tesla) and radiofrequency (rf) field strength of t1 G in the The most intense of these signals, near g = 2, was initially at- rotating frame. Microwave power and rf sweep rate varied, as tributed to copper (6) and is often referred to as the "intrinsic discussed below. EPR spectra simulations were calculated by copper" spectrum. Although this signal displays the charac- using program SIM 14 (17). teristic form of a cupric ion center (gII > go ge), it never- theless shows an unusually low value of gII, exhibits no hyperfine RESULTS splittings (hfs) from copper at X or Q band, has one principal- axis g value of less than 2.00, and has an anomalous tempera- EPR. In Fig. 1 the X-band EPR spectrum of the g = 2 region ture-dependent spin-lattice relaxation rate (7-9). Simulations of a frozen solution of cytochrome oxidase is shown. The signal of X-band EPR spectra are consistent with the presence of a from the "intrinsic Cu" site is free from features associated with Cu(II) ion (5), and Froncisz et al. (9) have recently reported the adventitiously bound copper (7) and, except for possibly higher resolution in.2- to 4-GHz EPR of hyperfine structure that may resolution brought about by the glycerol in the medium, is in be-from copper. However, it has also been noted that the spectra complete accord with the spectrum published by van Camp et features listed above are characteristic of thiyl radicals (R-S.) al. (11). Both these spectra (see Fig. i and ref. 11) are noticeably (10), and, in a recent electron-nuclear double resonance better resolved than the spectrum of Greenaway et al. (5), (ENDOR) investigation of cytochrome oxidase, proton and perhaps because of unfavorable conditions used by these au- nitrogen ENDOR was observed, but copper could not be de- thors, such as high microwave power, high modulation am- tected (11). plitude, and relatively high temperature. The superficial ap- To determine whether the metal ion is indeed part of this pearance is that of an axial g tensor (gII > g1), but Q-band oxidation-reduction center, we have reinvestigated the spectra show a rhombic splitting of the g1 region and give the ENDOR of cytochrome c oxidase. Although the EPR of cupric g values listed in Table 1 (3). As noted (10), the spectrum,.and complexes is.almost invariably straightforward, the observation in particular the g tensor, is at least as similar to that of a thiyl of ENDOR from the copper nucleus has been unaccountably radical (18-20) as it is to the type I copper centers with most difficult. We have recently studied the copper ENDOR of a similar g tensors (21). Representative g values for the blue number of blue copper proteins and found the proper condi- copper proteins and for a thiyl radical are also listed in Table tions for its detection (unpublished, and see refs. 12 and 13). The 1. present communication reports the copper ENDOR from the ENDOR. ENDOR is performed by inducing nuclear tran- intrinsic copper signal of cytochrome oxidase and presents a sitions with a rf field while observing the EPR signal intensity discussion of the electronic and geometric properties of this with the external magnetic field, Ho, set a fixed value (22). Field metal center. positions at the extreme edges of the EPR spectrum, near either g. or gx (positions A and C in Fig. 1) will give single crystal-like The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: ENDOR, electron-nuclear double resonance; EPR, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate electron paramagnetic resonance; hfs, hyperfine splittings; rf, radio- this fact. frequency. 1452 Downloaded by guest on September 27, 2021 Biophysics: Hoffman et al. Proc. Natl. Acad. Sci. USA 77 (1980) 1453 angle-dependent g value is equal to gy. Thus, patterns obtained at these fields are typically more poorly resolved and less sus- ceptible to interpretation. With Ho set near any of the positions indicated in Fig. 1, when the frequency of the rf field is swept slowly (dv/dt < 10 MHz/sec) and the microwave power is relatively low (<20 100 G gW), we observed ENDOR from protons and nitrogen.t Fig. I 2a presents the 'H and 14N signals taken at position B with a broad frequency sweep (70-3 MHz). An expansion of the region t For an individual set of protons, the ENDOR frequencies are v = VH + AH/2, in which VH is the free-proton Zeeman frequency (13.62 MHz at 3200 G) and AH is the hyperfine coupling. For 14N, in gen- B eral, a four-line pattern centered at AN/2 is anticipated (22). 'H 14N \C I FIG. 1. EPR spectra of cytochrome c oxidase. (a) Absorption- derivative spectrum (77 K, 50-mW microwave power, 5-G modulation amplitude). (b) Dispersion-derivative spectrum (2.0 K, 20-iW mi- crowave power, 4-G modulation amplitude). The letters A-C and arrows indicate positions where ENDOR spectra were taken (see text). patterns from molecules with the magnetic field directed along those g tensor axes (23). ENDOR at fields near B (Fig. 1) will produce signals from molecules with Ho along gy, but to these will be added the signals from all orientations at which the Table 1. EPR and ENDOR parameters for cytochrome c oxidase and possibly related speciesa Axis 60 40 20 Species x y z g values Cytochrome c oxidase 1.99 2.02 2.18 Type I (blue) copper 2.02-2.08 2.23-2.32 RCH2-S. 1.990 2.006 2.214b Copper hfs, MHz Cytochrome c oxidasec 68d 98e 90f Stellacyanin 167 87 96 63'65Cu Isotropic proton hfs, MHz Cytochrome c oxidaseg a,s, = 12.0 a,62= 19.1 RCH2-S- = 61.9 82.3 I aps a2= 1 20 80 40 a g values for type I copper, ref. 5; hfs for stellacyanin, ref. 12; data for MHz RCH2S- (electron-irradiated N-acetyl-L-cysteine) ref. 20; g values FIG. 2. ENDOR traces of cytochrome c oxidase taken at 2.0 K for cytochrome c oxidase, ref. 3. and showing peak assignments for 1H, 14N, and natural-abundance b Crystalline thiyl radicals also show a range, 2.21 _< gms < 2.29 (see 63 65Cu. Spectra a and b were taken at position B in Fig. 1. Spectrum references contained in refs. 18-20). a shows 1H and 14N ENJ)OR: 20-,uW microwave power, 4-G modu- c :15 MHz. lation amplitude, and rf scan rate 7 MHz/sec. The arrow indicates the d Limiting value of IACUI obtained at position C of Fig. 1 (see weakly coupled protons unresolved under these conditions. Spectrum text). b shows the emergence ofthe 63,'5Cu peak and the smearing of 1H and e High-frequency feature of Cu-ENDOR pattern at field B, Fig. 1 (see 14N signals under changed conditions: 200-,uW microwave power and text). rf scan rate-,160 MHz/sec. Spectrum c is the copper ENDOR spec- f IACUI obtained at field A (Fig.
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