Orientation and reactivity of cytochrome aa3 heme groups in proteoliposomes

ARCHIVESOF BIOCHEMISTRY AND BIOPHYSICS Vol. 204, No. 2, October 15, pp. 533-543, 1980

Orientation PETER

*Department fDepartment

and Reactivity of Cytochrome Groups in Proteoliposomes NICHOLLS,* AND JOHN

aa

Heme

VIRGINIA HILDEBRANDT,” M. WRIGGLESWORTH?

of’ Biological Sciences, Brock University, St. Catharines, Ontario L2S ~AI, Canada, and of Biochemistry, Chelsea College, Manresa Road, London SW3 6LX, United Kingdom Received

February

12, 1980

Reduction of cytochrome aa:, in proteoliposomes with ascorbate plus cytochrome c confirms that not more than 55% of the molecules are externally accessible and that the remainder are reduced only on the addition of membrane-permeable N,N,N’,N’tetramethyl-p-phenylenediamine. Reduction in the presence of terminal inhibitors such as cyanide, azide, and carbon monoxide shows that likewise 50% of the cytochrome a is accessible and 50% inaccessible. Dithionite reduces part of the cytochrome aB in the presence of azide, and none in the presence of cyanide. Methyl viologen, which is somewhat membrane permeable, can reduce part of the cyanide-complexed cytochrome a:, at low concentrations and all of it at high concentrations. Cytochrome aB is therefore also distributed randomly inside and outside the vesicles. Cytochrome c oxidase with externally facing cytochrome a is stimulated to high activity by its membrane association. Its turnover is dependent on the external pH and it is inhibited by external azide; trapping of azide cannot be used to demonstrate the orientation of the cytochrome a:, hemes associated with externally facing cytochrome a. Cytochrome c oxidase with internally facing cytochrome a is rather sluggishly reactive. Its low activity accounts for the apparent failure of detergents to release extra activity on lysing proteoliposomes. Double reciprocal plots of the reaction of added cytochrome c with proteoliposomes indicate apparent biphasic binding in the energized state, which is abolished upon the addition of uncouplers and valinomycin. But no transmembraneoua effect upon the oxidase reaction other than energization has been identified.

Natural membranes are not symmetrical. Both lipid distribution (1) and protein orientation (2) depend upon which side of the bilayer is “inside” (intracellular, or intraorganellar) and which “outside” (extracellular, or extraorganellar). Liposomes prepared artificially might be expected to be chemically symmetrical (3), but whether this is actually so is less clear (4). The smallest such vesicles have an internal diameter of about 160 A (5), and the radius of curvature of the two layers of lipid is very different. If an incorporated protein such as cytochrome c oxidase (6) could distinguish the two surfaces, its asymmetric assembly into the mitochondrial membrane would be less of a biosynthetic puzzle. Asymmetric incorporation of cytochrome c oxidase into artificial membranes would also help in studying its own mechanism of 533

action. Although the cytochrome a heme is accessible both to cytochrome c (7) and to calcium ions (8) from the outside of the inner mitochondrial membrane, it is not obvious from which side the cytochrome a, heme is accessible. Observations that indicated internal OH- generation from attack by O2 can also be interpreted by indirect mechanisms (9); and valinomycinstimulated azide inhibition (10) may likewise reflect indirect interactions rather than the postulated azide accumulation in the mitochondrial matrix (11). We have previously used proteoliposomes containing incorporated cytochrome c oxidase (6) to study the interactions between energization and the 0, reaction (12), between energization and ligand binding (13), and between energization and the spin state shift (14), as well as 0003.9861/80/120533-11$02.00/O Copyright B 1980 by Academic All

rights

of reproduction

in nny

Press, Inc. form

rcscrred.

534

NICHOLLS,

HILDEBRANDT,

vectorial kinetic properties (15). Such proteoliposomes prepared by cosonication of enzyme and phospholipid contain up to 50% of the cytochrome c oxidase inaccessible to membrane-impermeable reagents (16). This inaccessible enzyme also shows reduced catalytic activity, which complicates the use of such activity to probe orientation. Previous studies left uncertain the question as to whether both the hemes of cytochrome oxidase are equally inaccessible, and also whether if one heme on a single molecule is inaccessible the other heme is also inaccessible (the “transmembrane” problem). The present paper describes experiments using cytochrome a3 ligands to study heme orientation and trapped cytochrome c to examine reactivity of the inaccessible oxidase molecules. We employ the reagent described by Cox (17), methyl viologen, which is capable of reducing liganded heme groups, to reduce the accessible and inaccessible cytochrome a, groups when cyanide bound. And we attempt to use pH and azide concentration gradients to control the catalytic activity of the two kinds of cytochrome au3 in the proteoliposomes. A preliminary version of this paper was presented at the XI International Congress of Biochemistry in Toronto in 1979 (18). MATERIALS

AND METHODS

Three types of proteoliposome were employed: sonicated proteoliposomes, which were prepared essentially according to method (b) of Hansen et al. (12) or Wrigglesworth (15); using either acetone-washed asolectin (below) or purified phospholipids with PC:PE:DPG weight ratios of either 4:4:2 or 12:4:5; (ii) cholate-dialyzed proteoliposomes of purified phospholipids, which were obtained by dialyzing mixtures of cytochrome aaS, sonicated phospholipid plus cholate, according to Krab and Wikstrom (19); and (iii) vesicles with internally trapped cytochrome c, which may be prepared by either of the first two methods, carrying out the sonication or dialysis in the presence of 100 pM cytochrome c followed by passage through a 1.5 x lo-cm CM-Sephadex column either in 50 mM phosphate, pH 7.4 (Fig. 4), or in a medium identical with that used for assay (Table II). In all three types of proteoliposome the lipid: protein ratio was 50~1 (w/w).

(i)

AND WRIGGLESWORTH Cytochrome aa was prepared from beef heart according to Kuboyama et al. (20), and repurified by a modification of the method of Kessler et al. (21). In this latter procedure the enzyme is diluted to approx 10 mg.mll’ protein (40 pM cytochrome au,) and dialyzed for 48-72 h at 4°C against 25% ammonium sulfate, 2% cholate, and 0.1 M potassium phosphate, pH 7.4. The colorless precipitate is then discarded, and the solution dialyzed for a further 24 h against 0.1 M phosphate and minimal (~0.1%) amounts of Tween-80 to keep it in solution. Asolectin was obtained from Associated Concentrates Inc., Long Island, New York. Before use it was stirred for 3 h with a 5- 10 times excess of acetone, and then washed with fresh acetone on a Buchner filter funnel and dried. PC (phosphatidylcholine),’ prepared from egg yolks, was obtained from Peter Rand of the Department of Biological Sciences at Brock University. PE (phosphatidylethanolamine, from egg, Type I) and DPG (cardiolipin, beef, Type I) were obtained from Lipid Products Inc., Nutfield Nurseries, Redhill, Surrey. The cytochrome c used was Sigma Type VI from horse heart. Polylysine (M, approx 72,000) from Sigma Type IB. N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD), sodium ascorbate, and methyl viologen (MV+) were also Sigma products; Na,S,O, was from BDH Ltd., Poole, United Kingdom. Spectra were obtained with an Aminco DW-2 spectrophotometer in the split beam mode, and this instrument was also employed in the dual wavelength mode for the kinetic experiments. Rates of oxygen uptake were determined with a Clark electrode (Yellow Springs Instrument Co.), thermostatted at 30°C and attached to a suitable polarizing box and lomV recorder. Concentrations of cytochrome aa are expressed in terms of the presumed au3 unit with an extinction coefficient of 27 mM-’ for the reduced minus oxidized spectrum, 605-630 nm. As isolated the enzyme usually contained approximately 4 nmol au3 (8 neq heme a)/mg protein; after repurification (above) the concentration rose to about 5 nmol ua3 (10 meq heme a)/ mg protein. Protein concentration was determined by the biuret method. RESULTS

All three types of proteoliposome showed respiratory “control,” as measured polaroI Abbreviations used: TMPD, N,N,N’,N’-tetramethyl-p-phenylenediamine; MV+, reduced methyl viologen; PC, phosphatidyl choline; PE, phosphatidyl ethanolamine; DPG, diphosphatidyl glycerol (cardiolipin); MOPS, morpholinopropane sulfonate; FCCP, p-trifluoromethoxycarbonyl cyanide phenyl hydrazone.

ORIENTATION/REACTIVITY

OF CYTOCHROME

graphically by their response to addition of uncouplers and valinomycin (12), which increased respiration rates (in the presence of ascorbate, TMPD, and cytochrome c) up to sixfold with both asolectin and pure lipid systems. Vesicles with trapped cytochrome c (method (iii) above) showed the lowest control ratios when respiring on external cytochrome c. As reported previously (15, 16), after adding ascorbate and cytochrome c to vesicles prepared by method (i), and allowing anaerobiosis to supervene, about 48% reduction was seen at 445 nm and 55% at 605-630 nm, based upon 100% at each wavelength secured on addition of TMPD. Assuming that cytochromes a and a3 contribute equally to the absorbance change at 445-470 nm, but that cytochrome a contributes about 80% at 605-630 nm (ll), this is equivalent to 60% reduction of cytochrome a and 40% reduction of cytochrome u3. The addition of uncouplers and valinomycin is without significant effect on these reductions, either in rate or extent. Similar experiments were undertaken in the presence of ligands combining with the cytochrome u3 heme. Figure 1 compares the effects of cyanide, azide, and carbon monoxide on the behavior of liposomal cytochrome uu3. In the presence of cyanide, only cytochrome a can be reduced (22). Figure 1A shows that 55% of the liposomal cytochrome a is reduced by ascorbate plus cytochrome c. Slight changes in the apparent reduction over the first few minutes are due to the spin state shift in cytochrome u3 as cyanide forms its low-spin complex with the iron (22). Figure 1B illustrates the analogous results obtained in the presence of azide. Reduced cytochrome a hemes linked to u3Fe3+HN3 centers show a shifted (Y peak, distinct from that seen in the fully reduced enzyme or in the half-reduced cyanideinhibited system (11, 22). Due to this heme-heme interaction, it is therefore possible to distinguish between a mixture of equal parts of u3+ui+HN, and the corresponding mixture of u2+u~+ and u3”ag+HN3. Figure 1B shows the spectra of the ascorbate plus cytochrome c-reduced liposomal cytochrome uu3, as well as the

aa, HEME

GROUPS

535

ascorbate-TMPD-reduced liposomal cytochrome uu3. Both species show the characteristic blue-shifted 603-nm (Y peak of the cytochrome u2+uif+HN3 complex, although the reduction in the absence of TMPD is only 50% of that in its presence. The species produced in the presence of azide is therefore always uzfu~+HN3. The same species is produced on the addition of dithionite in the absence of cytochrome c and TMPD (not shown); external cytochrome a is accessible to dithionite, but neither external nor internal cytochrome u3 can be readily reduced by dithionite in the presence of NaN,. Similarly, carbon monoxide binds to the liposomal cytochrome au:, only after TMPD has been added to reduce internal a or u3 (Fig. 1C). Proteoliposomal cytochrome u is 57-60% reducible by dithionite, slightly more than by ascorbate plus cytochrome c. External cytochrome a reduction is accompanied initially by a spin state shift in the associated cytochrome u3 (22), seen here in the absence of cytochrome c (change from dashed to solid curve). Dithionite cannot react rapidly with either “external” or “internal” cytochrome a, hemes. Methyl viologen was introduced as a reductant of liganded heme proteins by Cox (17, 23). Figure 2 shows that unlike dithionite this reagent can reduce both free and liposomal cytochrome u3 even in the presence of quite high concentrations of cyanide, sufficient to ligate more than 99.9% of the available heme groups. About 60% of the TMPD-reducible heme in the proteoliposomes is reduced by dithionite alone (traces (b), (c), and (d)). The remaining cytochrome a heme is reduced by TMPD and the resulting half-reduced u”@HCN species can be fully reduced to u2+u~+HCN by adding methyl viologen. Methyl viologen is reduced to the blue radical form (Eh - -400 mV) by dithionite. Fortunately the 445- to 470-nm-wavelength pair is unaffected by this color, as the absorbance is identical at the two wavelengths. Addition of small amounts of methyl viologen to the free enzyme after TMPD (trace (a)) therefore induces reduction of all the a, hemes present. But addition of similar small amounts of MV+ to proteoliposomes is less effective

536

NICHOLLS,

HILDEBRANDT,

AND WRIGGLESWORTH

asalectin +Cyt.

4

a_a, -vesicles +3.3mM

CNA

o--a,

-

vesicles

(aralectin)

+0.73 UM E +4.6 mM NaN,

550

ao3 vesicles + carbon

(asolectin) monoxide

C (-ImM)

O’OZA

390

630 “rn

590

0’005A

430

470

570

610

653

nm

FIG. 1. Reducibility of liposomal cytochrome c oxidase in the presence of terminal inhibitors. Approximately 1 /AM liposomal cytochrome aa3, in the form of asolectin vesicles with cytochrome c added externally, was reduced in semimicro cuvettes (final volume 1.1 ml) in 50 mM sodium phosphate buffer, pH 7.4, at 30°C. (A) Effect of 3.3 mM KCN. Spectra were obtained upon the addition of 10 pmol ascorbate, initially (- - -) and finally (--), and 0.45 pmol TMPD (- -), in the presence of 0.2 pM cytochrome c. (B) Effect of 4.6 mM NaN,. Spectra were obtained upon the addition of 10 pM ascorbate (- - -), 0.45 pmol TMPD (- -), and dithionite (-), in the presence of 0.7 pM cytochrome c. (C) Effect of CO (bubbled at atmospheric pressure, (-0.9 mM). Spectra were obtained upon the addition of dithionite, initially (- - -) and finally (---), and of 0.45 pmol TMPD (- -).

ORIENTATION/REACTIVITY

OF CYTOCHROME

(~(1%HEME GROUPS

537

FIG. 2. Kinetics of reduction of cytochrome au3 as isolated and in asolectin vesicles, measured at 445-470 nm in the presence of cyanide. Cytochrome au,-containing vesicles, as in Fig. 1, were suspended in 50 mM phosphate, pH 7.2, 3O”C, and incubated for 48 h in the presence of 1 mM KCN. (a), Control, showing reducibility of free enzyme by dithionite, TMPD, and methyl viologen; (b), (c), and (d), proteoliposomes, showing reducibility by dithionite, TMPD, and low and high levels of methyl viologen; 1.6 PM cytochrome uus, as isolated or as asolectin vesicles (type i) was used in a total volume of 1.1 ml.

(traces (b), (c), and (d)). Part of the ag+HCN is reducible by small amounts of MV+, while the remainder requires a higher eoncentration (trace (d)). Despite its size and presumed positive charge when reduced, methyl viologen is somewhat membrane permeable. Using cytochrome c trapped in hand-shaken liposomes (the Kimelberg and Lee (24) experiment), methyl viologen will act like TMPD in overcoming the permeability barrier to dithionite (not shown). As illustrated in Fig. 3, the cyanide complex of cytochrome a3 in intact beef heart mitochondria is reducible by Na,S,04-MV+, at least when the methyl viologen eoncentration is raised. Yet this cannot be interpreted as indicating an external location of the heme upon the membrane. Proteoliposomes made by sonicating cytochrome aa with purified phospholipids (see under Materials and Methods) show similar behavior to that of the asolectin vesicles. Dithionite can reduce up to 60% of the total heme, and in the presence of cyanide, up to 65% of the available cytochrome a. Cholate-dialyzed vesicles (method (ii) above) behave in essentially the same way, although in these vesicles up to 75% of the heme (a + a,) may be reduced by ascorbate plus external cytochrome c. These vesicles may therefore be somewhat more asymmetric than the

sonicated ones, as previously proposed by other workers (25). The differences between the two types of proteoliposome will require further study.

I

~

I -\ ~ STEM CN- /

sucrose-monn,tol-MOPS~EDTA pH 74 30°C a

FIG. 3. Reducibility of mitochondrial cytochrome by methyl viologen and dithionite in the presence of cyanide. BHMw, indicates the reduction seen in beef heart mitochondria, approx 2 mg’mll’ (protein); uu3, indicates the behavior of a control containing 1.1 pM isolated enzyme. The medium used was 225 mM mannitol, 75 mM sucrose, 10 mM (Na) MOPS, 1 mM EDTA, at pH 7.4, 30°C. Reduction was monitored at 445-470 nm (dual wavelength) as in Fig. 2. aa,-,

538

NICHOLLS,

HILDEBRANDT,

AND WRIGGLESWORTH

FIG. 4. Difference spectra of cytochrome c-loaded proteoliposomes. Cytochrome aa,-containing sonicated proteoliposomes were prepared with asolectin in the presence of cytochrome c (method (iii)) and passed through a CM-Sephadex column as described under Materials and Methods. Samples were diluted in 50 mM Na phosphate buffer at 30°C and placed in two cuvettes. Ascorbate (7.0 mM) was added to the sample cuvette and anaerobiosis allowed to occur: a second addition of TMPD (0.18 mM) was then made to secure full reduction. - - -, Oxidized vs oxidized baseline (0.5 A full scale in Soret region, 0.1 A in visible region); - -, ascorbate-induced anaerobic spectrum (0.5 A full scale in Soret region, 0.1 A in visible region); -, TMPD-induced full reduction (0.5 A full scale in Soret region and in visible region to 580 nm, 0.2 A full scale above 580 nm).

Sonicated proteoliposomes with internally trapped cytochrome c, prepared by method (iii), show a 50:50 distribution of externally and internally facing cytochrome ao3 units (Fig. 4), just like vesicles sonicated in the absence of cytochrome c. Vesicles prepared in 50-200 PM cytochrome c solutions and then passed through a cationexchange column, as described under Materials and Methods, contain between 5 and 10 times as much cytochrome c as cytochrome uu3. Nearly all this trapped cytochrome c is reduced only in the presence of TMPD (24). Only 10% of the cytochrome c (approximately equimolar with the cytochrome au& is ascorbate reducible, as shown in Fig. 4. Evidently the cation-exchange process removes cytochrome c in solution and all of that bound nonspecifically; a small quantity associated with cytochrome c oxidase remains ascorbate accessible, directly or indirectly. These cytochrome c-containing vesicles could be used to study the activity of both internally and externally facing cytochromeuu, molecules, in the presence and absence of inhibitors such as azide, Palmieri and Klingenberg (10)

had suggested that mitochondria may be loaded with azide and that under these conditions cytochrome c oxidase is very strongly inhibited. It was concluded that mitochondrial cytochrome u3 responds to ligand concentration in the matrix. However, most of their data were acquired in the presence of valinomycin, which complicates the analysis of cytochrome c oxidase activity (11, 26). An attempt was therefore made to study azide binding kinetically in proteoliposomes by “loading” the vesicles. The results obtained showed that the reactivity of liposomal cytochrome au3 (asolectin proteoliposomes) with externally added cytochrome c is unaffected by such prior loading of the vesicles with inhibitor. Azideloaded vesicles (internal pH 6.7) behaved identically to control vesicles toward exogenous cytochrome c, in an external medium pH 7.4, both in presence and in absence of FCCP and valinomycin. This implies that either azide is too rapidly permeable (even when loaded at a low pH) for an inhibitory effect to be demonstrable or that externally reactive oxidase is af-

ORIENTATION/REACTIVITY

OF

fected only by external azide. Higher vesicle concentrations were then employed containing trapped cytochrome c in the presence of external polylysine to measure the activity of internally facing enzyme. Such respiration was also not especially sensitive to internal azide. We conclude that azide permeability is too great for effective trapping; this was true even though a pH gradient was also superimposed upon the system. Some of the findings of Palmieri and Klingenberg (lo), which we were unable to repeat with mitochondria (ll), thus cannot be mimicked with proteoliposomes. There are, however, some differences between internally and externally facing oxidase in azide sensitivity; as the maximal activity of internally facing oxidase is lower, so is its azide sensitivity. If conditions inside the vesicle diminish the enzyme’s sensitivity to azide, this effect may counteract any effect produced by azide loading. The kinetic behavior of the outwardfacing oxidase is normally controlled by conditions in the external and not the intraliposomal medium. Wrigglesworth (15) and Petersen (27) have concluded that only external pH affects the vesicular enzyme, TABLE

I

MAXIMUM TURNOVER NUMBERS OF LIPOSOMAL CYTOCHROME a~l.,~ PH 7.2 7.4 7.85

TNbpp)” 300 s- I 186 s-’ 125 s-1

TN(max)h

TN(exo)“

464 s-1 287 s-1 193 s-1

773 s-f 479 s-‘1’ 322 s-’

Note. 50 mM potassium (pH 7.2) or sodium (pH 7.4 and 7.85) phosphate buffer, 3O”C, approx 7 mM ascorbate, 200 PM TMPD, with 12.5 nM liposomal cytochrome aaB, in the presence of valinomycin and FCCP as in Fig. 5: “respiratory control” ratios between 4 and 5; 40% PC:40% PE:20% DPG phospholipid vesicles (cf. Materials and Methods). ” At 11 ~$1 cytochrome c (standard assay), in electronsisecondlcytochrome aa,,. ” Extrapolated to infinite [cl (K,,, = 6 PM) (cf. Ref. 12 and Fig. 5). ” Assuming that no more than 60% of the total cytochrome cm:,is catalytically active (this paper). ” cf. values for mitochondria (400-550 s-l) and submitochondrial particles (300-400 SK’) in Ref. (28).

CYTOCHROME

aa3 HEME TABLE

539

GROUPS II

ACTIVITY OF OUTWARD- AND INWARD-FACING OXIDASE MOLECULES Respiration rate (pM 0, min-I) Type of vesicle

Additions

A. Sonicated None (method FCCP and valinomycin (i + iii)) B. Dialyzed (method (ii + iii))

None FCCP and valinomycin

(a) Total

(b) Internal

(a)-(b) External

51

12

39

89

15

74

28

7

21

49

6

43

Note. Medium contained: (A) 75 I!IM sucrose, 225 mM mannitol, 10 mM MOPS, 1 rn~ EDTA, pH 7.4, 30°C. (B) 220 mM sucrose, 5 mM MOPS, 5 IIIM Tris, pH 7.4, 30°C. Son&ted vesicles(A) contained 9.4 q~~l trapped cytochromeci pm01 cytochrome aa, (total). The oxygen electrode system contained 38 nM liposomal aag. Dialyzed vesicles(B) contained 7.3 pm01 trapped cytochrome ci~mol cytochrome sag (total). The oxygen electrode system contained 15 nM liposomal an,. The reagents included 7 mM ascorbate, 0.18 ITIM TMPD, 12-14 FM q&chrome E (column (a)), 1.2 FM FCCP, 1.2 pg.ml-’ valinomyein, 48 ,u*g.mlk’polylysine (column (b)).

either in the presence or absence of uncouplers, and that the resulting pH curve is similar to that for isolated (nonvesicular or micellar) enzyme. This applies both to overall turnover (15) and to reaction with 0, (27). Variations in internal pH (15, 16) are much less important. Table I lists turnover numbers obtained in our experiments for externally facing cytochrome c oxidase, extrapolated to infinite cytochrome c concentration. The numbers obtained are equal in magnitude to those calculated for mitochondrial cytochrome c oxidase, and rather higher than those shown by isolated enzyme activated by detergents (28). When proteoliposomes containing cytochrome c oxidase are loaded with cytochrome c (method (iii)), however, the turnover numbers obtained for the “inward-facing” oxidase are much lower, as indicated by the results in Table II. These data show that in vesicles where the oxidase is topologically “scrambled” by the spectroscopic criteria (Fig. 4), the measured activity of “internal” oxidase, obtained in the presence of polylysine is much lower than that of “external” enzyme, especially in the pres-

540

NICHOLLS,

HILDEBRANDT,

AND WRIGGLESWORTH

about 6 PM (Fig. 5, closed symbols). In the absence of ionophores (Fig. 5, open symbols), biphasic plots are seen, with cytochrome c showing a similar K, to that in the deenergized state at high concentrations, but a lower K,n at low concentrations. This feature is discussed below. DISCUSSION

FIG. 5. Double reciprocal plot of cytochrome c oxidase activity in sonicated (asolectin) proteoliposomes, Proteoliposomes were made according to method (i) (Materials and Methods) and assayed polarographically in the presence of varying levels of cytochrome c using 7 mM ascorbate and 175 pM TMPD as substrate. Open symbols (0, 0) indicate the control, unstimulated, rates. Closed symbols (0, n ) indicate the rates in presence of 1.2 /.LM FCCP and 0.12 wg.rnl-’ valinomycin. The medium was 50 mM phosphate, pH 7.45, at 30°C in 4.3 ml total volume. A fivefold higher concentration of vesicles (0, 9) was used at the lower cytochrome c concentrations.

ence of FCCP and valinomycin. These reagents have little stimulatory effect on the activity assigned to the internal enzyme. Spectrophotometric studies (not shown) indicate that endogenous cytochrome c is almost completely reduced in the steady state under these conditions. The low turnover must therefore reflect either a decreased intrinsic turnover of the ferrocytochrome c enzyme complex or a poor mutual accessibility of internally facing enzyme, and trapped cytochrome c, despite the latter’s putatively high concentration. Figure 5 shows that the externally facing enzyme displays a “normal” affinity for cytochrome c in the deenergized state, and a single K, value in ionic media (for reviews see Refs. 29 and 30). In the presence of FCCP and valinomycin this K, value is

Figure 6 illustrates the two models of membrane-bound cytochrome c oxidasethat advocated by Moyle and Mitchell (31), the transmembrane model, and that advocated by Wikstrom and Saari (32), the linked proton pump model. In either case, the oxidase molecules may be distributed randomly (i.e., approximately 50% inward facing and 50% outward facing) or with some degree of preference for one orientation, as suggested by Eytan et al. (25). We have previously shown (16) that not more than 60% of the total incorporated cytochrome c oxidase can be reduced by Dlthlonlte

FIG. 6. Two models of the cytochrome aa,-containing proteoliposome. The upper transmembrane model is of the conventional “plugged through” Mitchell (9) variety, but with two orientations of cytochrome ou3, each partially accessible to reagents in the external medium. The lower “linked proton pump” model is of the Wikstrom and Saari (32) type, with two populations of cytochromeaa, molecules, some entirely inside and inaccessible, and the remainder fully exposed and accessible. Cytochromes n and a:, are indicated purely schematically in this figure and their relative positions show apparent accessibility to reducing agents rather than mutual distances or membrane location.

ORIENTATION/REACTIVITY

OF CYTOCHROME

membrane-impermeable reagents. The present results extend this to the two individual heme groups. Thus, when tested with ascorbate plus cytochrome c in the presence of a terminal inhibitor, 50% of cytochrome a groups in sonicated proteoliposomes are reducible; the remainder can only be reached on addition of membrane-permeable TMPD. Similarly, only some of the cytochrome a3 groups are directly accessible to Na,S,O, (Figs. 2 and 4); the remainder require addition of membrane-permeable methyl viologen, a reagent capable of reducing cyanoferricytochrome cc8 to the corresponding ferro form (17, 23). However, a decision as to whether the accessible cytochrome a groups are associated with inaccessible cytochrome a3 groups and vice versa (31), or whether the hemes on one molecule are either both accessible or both inaccessible (32), is harder to achieve. Chemical orientation cannot be determined by reagents indicating proximity to either membrane face. Although it is likely that the two hemes are close together, and that both may be nearer the C face of the inner membrane than the M face, it is the “vectorial” behavior of attacking and leaving species from the aqueous phases that is important. The catalytically active “externally accessible” cytochrome c oxidase cannot be inhibited by trapped azide, but this is probably because the latter diffuses out rapidly. Its reducibility by dithionite is somewhat diminished as compared with isolated enzyme; initial reduction is of only 25% total heme (33). This could be due to inaccessibility of the a3 heme; this possibility is supported by the persistence of the azide-induced a-peak shift of ferrocytochrome a in the presence of dithionite (Fig. 1B). On the other hand, the vesicular enzyme seems only to sense external pH and not internal pH (15, 16, 27), suggesting that the cytochrome u3 moiety functional in the oxidation of external ferrocytochrome c is itself external. All presently available cytochrome a, inhibitors other than H+ and OH- ions are membrane permeable, and therefore the orientation of cytochrome a, associated with externally facing cytochrome u cannot be directly ascertained

au:, HEME

GROUPS

541

using such a ligand. Internally facing cytowith chrome uu3, capable of interacting trapped cytochrome c, is different in its behavior. Its turnover is only 20% that of externally facing cytochrome uu3, reacting with added cytochrome c (Table II), and its azide sensitivity is also less. The latter property is shared with part of the cytochrome c oxidase in submitochondrial particles (34). The decreased reactivity of internally facing enzyme, coupled with the (‘viscotropic” (35) activation of externally facing enzyme, probably accounts for the failure of vesicle lysis to increase observed activity (16, 25). A more recent claim for preferential outward-facing incorporation of cytochrome oxidase into proteoliposomes was made by Casey et al. (36), on the basis of spectrophotometric steady-state studies, but appears to involve the doubtful assumption that in their system TMPD cannot reduce internal oxidase in the absence of cytochrome c, a conclusion which is contrary to our findings (cf. 22, 37). Whether or not both hemes of externally facing cytochrome au3 are externally accessible, such molecules clearly catalyze transmembraneous processes (15, 16). In the absence of ionophores internal alkalinization of the vesicles is occurring (15); under these conditions energization effects are also seen which modify the kinetic behavior (Fig. 5). Hansen et al. (12) concluded that “energization acts non-competitively towards cytochrome c oxidation.” That means that V,,,,, but not K,,, for cytochrome c should be affected by uncouplers and valinomycin. But on repeating such experiments over a wide concentration range it was found that a low cytochrome c concentrations, respiratory “control” values decline, indicating “uncompetitive” behavior of energization toward cytochrome c (Fig. 5). At higher cytochrome c concentrations, respiratory control reaches a maximal value. This latter is the region in which energization is acting “noncompetitively” toward cytochrome c. It is tempting to speculate that at low concentrations cytochrome c binding may be affected directly by membrane energization (membrane potential), unlike the reaction with oxygen (12), or that the two

542

NICHOLLS,

HILDEBRANDT,

binding sites for cytochrome c on cytochrome c oxidase (30) are not equally sensitive to membrane potential (i.e., one is intrinsically less “coupled” than the other). Explanations of less mechanistic interest are, however, also possible. Thus high cytochrome c concentrations may slightly deenergize the system by increasing ionic permeabilities. Although a number of lines of evidence indicate that cytochrome c oxidase is arranged transmembraneously, and although when incorporated into liposomes it can orient itself with cytochrome a facing inward or outward, it is uncertain whether the two cytochromes are transmembraneous or whether electrons traverse the membrane. Membrane energization influences cytochrome c binding (this paper) and possibly the oxygen reaction (38), although indirect kinetic effects may be responsible for the latter phenomena (12, 39). The results presented here and by others (16, 27) suggest that it is external conditions of pH, ionic strength, and probably inhibitor concentration that modulate externally facing oxidase molecules. Artzatbanov et al. (40) have reported that intramitochondrial pH determines the redox potential shown by cytochrome a in the presence of cyanide. Their results were obtained by pulsing potentiometrically poised mitochondrial suspensions with KOH or HCl solutions and showing that subsequent changes in the reduction level at 605-630 nm were accelerated on adding uncoupler and valinomycin. These effects may reflect interactions between the oxidoreduction of cytochrome a and the operation of transmembraneous proton channels; certainly membrane energization effects cannot be excluded as contributing factors in systems subjected to imposed pH gradients in the absence of uncouplers. A topochemically transmembraneous modification of the oxidase reaction, unrelated to any possible membrane energization, has yet to be found. ACKNOWLEDGMENTS This work was supported by NSERC Grant A0412 to P.N. and by travel grants from the Wellcome

AND WRIGGLESWORTH Foundation and from NATO to J.M.W. We thank Mrs. Freda Nicholls for assistance in preparing and testing proteoliposomes, and Mrs. Nola Fuller for the preparation of egg lecithin (phosphatidylcholine) used. We acknowledge discussions with cand. sci. Finn B. Hansen (State University of New York at Buffalo) and with Dr. Lars Chr. Petersen (Hvidovre Hospital, Denmark), and also thank the latter for a preprint of his paper on pH effects (Ref. 27). REFERENCES 1. BRETSCHER, M. S. (19’78) in Membrane Proteins (11th FEBS meeting Proceedings; Nicholls, P., Meller, J. V., Jorgensen, P. L., and Moody, A. J., eds.), Vol 45, pp. 13-16, Pergamon, Long Island City, N. Y. 2. KIMELBERG, H. K. (19’76) Mol. Cell. Biochem. 10, 171-190. 3. BANGHAM, A. D. (1972) Annu. Rev. Biochem. 41, 753-776. 4. JOHNSON, S. (1973) Biochim. Biophys. Acta 307, 27-41. 5. JOHNSON, S. M., BANGHAM, A. D., HILL, M. W., AND KORN, E. D. (1971) Biochim. Biophys. Acta

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