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Cytochrome c interaction with membranes
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Cytochrome
169, 199-208 (I9751
c interaction
with
Formylated
Cytochrome
MARIA
ERECIfiSKA
University
of Pennsylvania,
Johnson Research Foundation,
Received March
Membranes c’
Philadelphia,
Pennsylvania
19174
1, 1975
A single species of tryptophan-59 formylated cytochrome c with a half-reduction potential of 0.085 * 0.01 V at pH 7.0 was used to study its catalytic and functional properties. The spectral properties of the modified cytochrome show that the 6th ligand position is open to reaction with azide, cyanide, and carbon monoxide. Formylated cytochrome c binds to cytochrome c depleted rat liver and pigeon heart mitochondria with the precise stoichiometry of two modified cytochrome c molecules per molecule of cytochrome a (K, of approx 0.1 PM). Formylated cytochrome c was reducible by ascorbate and was readily oxidized by cytochrome c oxidase. The apparent K, value of the oxidase for the formylated cytochrome c was six times higher than for the native cytochrome and the apparent V was smaller. Formylated cytochrome c does not restore the oxygen uptake in C-depleted mitochondria but inhibits, in a competitive manner, the oxygen uptake induced by the addition of native cytochrome c. Formylated cytochrome c was inactive in the reaction with mitochondrial NADH-cytochrome c reductase but was able to accept electrons through the microsomal NADPH-cytochrome c reductase.
Horse heart cytochrome c contains a single tryptophan residue at position 59 of its polypeptide chain. This tryptophan residue is situated in close proximity to the heme and to two other aromatic residues, those of tyrosines 67 and 74, in what appears on the X-ray structure as the left “hydrophobic channel” (1). The relative positions of the three aromatic residues to each other and to the heme are dependent on the redox state of cytochrome c. In the oxidized molecule (1) the plape of tryptophan 59 is approximately 5 A away from the heme plane and parallel to tyrosine 74. Upon reduction, tryptophan rotates against the heme so that the alignment between its ring and that of tyrosine 74 is broken and assumes a new position in which it becomes parallel to tyrosine 67 (2). Because of the ease with which the H electron systems of the aromatic amino acid residues share their electron “clouds,” ‘Fifth paper in the series. Papers I-IV 11-14. Supported by USPH GM 12202-10.
are Refs. 199
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
Winfield (3) put forward a suggestion that tyrosine and tryptophan radicals were involved in electron transfer through the protein network of hemeproteins. The existence of the left “hydrophobic channel” filled with aromatic residues and their particular influence upon the redox reactions of cytochrome c led Dickerson and co-workers (1, 2) to postulate that the “conduction mechanism” was responsible for the electron transfer through the cytochrome c molecule. Supporting evidence for such a mechanism of electron transfer may be provided by the studies on cytochrome c derivatives in which the abovementioned aromatic residues had been individually modified. The tacit assumptions are: 1. That such modification influences only the amino acid in question, and 2. That modification affects either the rate of electron donation or withdrawal or both. In 1967, Previero et al. (4) showed that tryptophan reacts quantitatively in a solution of formic acid and HCl to give only
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ERECIhSKA
one product in which the formyl group is linked to the nitrogen of the indole ring. Aviram and Schejter (5) used this procedure to formylate the tryptophan residue of cytochrome c. In the modified cytochrome, they noted in addition to the disappearance of the 280 nm absorbance, the absence of the 695 nm absorbance band in the oxidized molecule, characteristic of the intact methionine-80 bond (6-8). The absence of the upfield-shifted resonances corresponding to protons of the Fe-bound methionyl residue in formylated cytochrome c (9) was considered evidence for the confirmation of the results of Aviram and Schejter (5). The formylated cytochrome c was autooxidizable and bound CO; it could not be reduced by ascorbate and was inactive in restoring the respiration in cytochrome c-depleted mitochondria (5). On the other hand, Margoliash et al. (10) reported that formylated cytochrome c was equally as active as the native molecule in the reaction with cytochorme c oxidase, although it was inactive in the reaction with cytochrome c reductase. In continuation of our previous studies on cytochrome c interactions with membranes (ll-14), we decided to look more systematically at the reactions of formylated cytochrome c. The starting point of our investigation was the intriguing observation (10) that TMPD [E,,.,, = +260 mV and 30°C (15)], but not ascorbate [E,,,, = +45 mV (16)], was able to reduce formylated cytochrome c. The questions we asked were: 1. What are the thermodynamic and binding characteristics of formylated cytochrome c? 2. How much can be learned from studies on modified cytochromes c of the natural electron transfer pathways? This paper reports some information relevant to these two questions. A preliminary account of the work has already appeared (17). MATERIALS
AND
METHODS
Materials Formylated cytochrome c was prepared according to the method of Aviram and Schejter (5). After dialysis of the reacted cytochrome c against 10 mM phosphate buffer pH 7.2, as described in the original
method, the solution of diluted cytochrome c was adsorbed onto a small CM-cellulose column and the final concentrated formylated cytochrome c was eluted with 0.4 M phosphate buffer pH 7.4. Cytochrome c oxidase w-as isolated from pigeon breast mitochondria either by the method of Kuboyama et al. (18) or by the method of Sun et al. (19). Microsomal NADP-cytochrome c reductase was solubilized from rat liver microsomes by controlled digestion with trypsin as described by Omura and Takesue (20) and used without further purification. Rat liver cytochrome c-depleted mitochondria were prepared by the method of Jacobs and Sanadi (21), and pigeon heart depleted membranes by the modification of this method described by Boveris et al. (22). Methods Oxidation-reduction potential measurements. The potentiometric titrations of formylated cytochrome c were carried out by using simultaneous measurements of absorbance and oxidation-reduction potential (for details see Ref. 23). Potassium ferricyanide was used as an oxidant and freshly prepared sodium dithionite solution as a reductant. The redox mediators used were: diaminodurene (Aldrich Chem. Co., Milwaukee, WI), phenazine methosulfate (Sigma Chem. Co., St. Louis, MO), phenazine ethosulfate (K & K, Plainview, NY), duroquinone (Aldrich), 2-hydroxynaphthoquinone (K & K) and pyocyanine (K & K); their concentrations were lo-15 pM. Measurements were carried out at 22-25°C. Binding of cytochromes c was determined as the amount of native or formylated cytochrome c found in the mitochondrial pellet after centrifuging down the cytochrome c depleted mitochondria (10 min at SoOOg) from the suspending medium (0.20 M sucrose-0.05 M morpholinopropane sulfonate buffer pH 7.0). The pellet was suspended in 0.1 M phosphate buffer pH 7.2 containing 1% Triton X-100. The concentration of cytochrome c was estimated from the difference between total oxidized (+ 5 mM ferricyanide)-total reduced (t dithionite) measured at 550-540 nm. The extinction coefficients used were 19.7 for native cytochrome c and 13.4 for formylated cytochrome c. The concentration of free cytochrome c was determined by measuring the amount found in the supernatant from the 8000g centrifugation. Oxygen uptake was measured at 24°C with a Clark oxygen electrode in 0.20 M sucrose-O.050 M morpholinopropane sulfonate pH 7.2 buffer, in the presence of succinate plus glutamate as substrate. Cytochrome oxidase activity was determined by measuring the rates of oxygen uptake at 24°C with a Clark oxygen electrode. Parallel experiments were carried out for the native and formylated cytochrome c. The results were corrected for the autoxidation rates of cytochromes c. The detailed conditions are given in the figure legends.
FORMYLATED NADPH-cytochrome c reductase activity was measured spectrophotometrically by following the rate of cytochrome c reduction (or formylated cytochrome c) at 550 nm. The assay medium contained: 2 x 10m5M cytochrome c, 1 x lo-’ M NADPH, and enzyme in 0.1 M phosphate buffer, pH 7.2. Total volume was 3 ml. The reaction was started by the addition of NADPH. Cyanide was omitted from the reaction mixture since it reacted with formylated ferricytochrome c. It was found that the rates of enzymatic oxidation of ferrocytochrome c were negligible, because partially purified NADPH-cytochrome c reductase was used for assays. Cytochrome a concentration was measured at 605-630 nm (reduced-oxidized) using an extinction coefficient of 24. Protein was determined by the biuret method (24). RESULTS
Spectral Characteristics of Formylated Cytochrome c
CYTOCHROME
201
c
28 Ii I’
24
I i
,20 16
E .I 2 -8
520
540 560
580
X (nm)
FIG. 1. Light absorption spectrum of horse heart cytochrome c (broken line) and its formylated derivative (solid line). Cytochrome c (or formylated cytochrome c) was suspended in 0.1 M phosphate buffer pH 7.0 at a concentration of 15-20 fiM.
The spectral characteristics of oxidized formylated cytochrome c have been described by Aviram and Schejter (5) and can be summarized as follows: 1. Disappearance of the 280 nm absorbance peak and simultaneous increase in absorbance at around 298 nm; 2. 3-4 nm shift in the Soret maximum toward shorter wavelength; 3. disappearance of the 695 nm 360 420 460 500520 560 6ocl 640 absorbance band. In addition to the Wovelengfh , nmI Wwelengfh I “In I changes in the spectrum of the oxidized FIG. 2. Absorption spectra of formylated cytomolecule, we have noted that the extincchrome c and its cyano and azide compounds. Oxition coefficients for the reduced absorption dized formylated cytochrome c was suspended in 0.1 bands of formylated cytochrome c differ M phosphate buffer pH 7.0 at a concentration of 16.5 from those for the native molecule: the pM (-) formylated cytochrome c; (-----) 15 mM extinction coefficient at 550 nm for the sodium azide; (- -. ~. ) 15 mM potassium cyanide. reduced cytochrome decreases from 27.9 (native cytochrome c) to 21.3 (formylated mylated cytochrome c has its (Y maximum cytochrome c). The calculations based on at 536 nm and Soret maximum at 415 nm; the data of Fig. 1 yield a value of 13.4 for the azide formylated cytochrome c peaks reduced minus oxidized extinction coeffiare at 537 and 412 for the (Y and Soret cient at 550 minus 540 nm for formylated maximums, respectively (the formylated cytochrome c. The Soret maximum of the cytochrome c exhibited peaks at 528 and reduced cytochrome is shifted 2-3 nm t.o- 405 nm). The reduced form of formylated ward the red and the extinction coefficient cytochrome c reacts with carbon monoxide is increased by -10% upon formylation. It as manifested by the decrease in absorpshould be pointed out that similar changes tion in the (Y region and an increase in the in the cytochrome c molecule were ob- Soret region. The Soret maximum of the CO compound of formylated ferrocytoserved by Yonetani (25) when it was modichrome c is at 416 nm. fied by N-bromosuccinamide addition. The spectrum of oxidized formylated Oxidized formylated cytochrome c reacts cytochrome c is pH dependent (Fig. 3). readily at neutral pH with added ligands, Lowering the pH causes the appearance of cyanide, and azide (Fig. 2). The cyanofor-
202
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ERECIfVSKA
(data not shown) and was essentially identical of the data of Wfithrich et al. (9). Thermodynamic
500
550
600
650
.Ahd FIG. 3. Effect of pH on absorption spectra of formylated cytochrome c. The buffers used were mixtures of 0.1 M citric acid and 0.2 M K,HPO, of appropriate pH. 17.8 FM formylated cytochrome c.
0:I
I Ox/Red
I'0
FIG. 4. Potentiometric titration curve of formylated cytochrome c. Formylated cytochrome c was suspended at a concentration of 5 pM in 0.2 M sucrose-O.050 M morpholinopropane sulfonate pH 7.0 buffer. The redox mediators used were: 20 pM diaminodurene, 40 pM each of phenazine methosulfate, phenazine ethosulfate and duroquinone, 5 FM pyocyanine, and 15 PM 2-OH, 1,4 naphtoquinone (0) oxidative titration with potassium ferricyanide; (A) reductive titration with sodium dithionite.
an absorption peak at 618-620 nm, characteristic of high spin compounds. Below pH 6.5 formylated cytochrome c is a mixture of high and low spin forms. The latter information is confirmed by EPR spectroscopy which shows a gradual increase in the peak with a g value of 6.0 when the pH is lowered (not shown). The NMR spectrum of oxidized formylated cytochrome c showed the absence of the upfield shifted resonances at 3.7, 3.3, and 2.7 ppm corresponding to the protons of the iron-bound methionyl-80 residue
Properties of Formylated Cytochrome c
Anaerobic potentiometric titrations of formylated cytochrome c prepared as described by Aviram and Schejter (5) yield a sigmoid titration curve, characteristic of two components (Fig. 4). Resolution of this titration curve into the two components gives the values of their half-reduction potentials: 0.090 f 0.01 V for the higher potential one and -0.055 V for the lower. Both components titrate with a slope of 1 which indicates that they are one-electron donors/acceptors. When formylated cytochrome c, purified on a small CM-cellulose column is titrated under the same conditions only one component is observed with a half-reduction potential of 0.085 V (i.e., it corresponds to the high potential component of the nonpurified formylated cytochrome c) (Fig. 5). All of the studies described below were done with formylated cytochrome c with an E,,.O of 0.085 V. Treatment of formylated cytochrome c at alkaline pH (5) causes displacement of the formyl group and return of the spectral properties to those of the native form. The thermodynamic properties of formylated cytochrome c also revert to their original value; the midpoint potential of .
FIG. 5. Potentiometric titration of CM-cellulose purified formylated and deformylated cytochrome c. Deformylation was carried out by alkaline treatment as described by Aviram and Schejter (1969) 5.1 FM formylated cytochrome c, 4.8 PM deformylated cytochrome c. Conditions are those of Fig. 5.
FORMYLATED
CYTOCHROME
c
203
FIG. 6. Binding of native and formylated cytochromes c to cytochrome c-depleted rat liver and pigeon heart mitochondria. Cytochrome c depleted mitochondria (at least 1 pM cytochrome a) were incubated in 0.20 M sucrose-O.050 M morpholinopropane sulfonate pH 7.0 buffer with various amounts of cytochromes c for 5 min at room temperature. The buffer was presaturated with oxygen and the samples aerated during the incubation time. The mitochondria were removed by centrifugation at 8OOOg for 10 min at 4°C. The pellet was suspended in 0.1 M phosphate buffer pH 7.2 containing 1% triton X-100. The concentration of cytochromes c in the pellet and in the supernate was determined as described in Methods; (0, 0) horse heart cytochrome C; (A, A) formylated horse heart cytochrome c.
“deformylated” cytochrome c was found to be indistinguishable from that for the native molecule (Fig. 5). Attempts to measure the half-reduction potential of the formylated cytochrome c in the presence of negatively charged phospholipid vesicles were rather unsuccessful. The molecule appeared to be labile-a fraction of the 0.085 V component was converted into a form which was very difficult to reduce which suggested to us possible denaturation of the formylated cytochrome c. Since a relatively high concentration of cardiolipin was used in these experiments, the local density of negative charges may have been very high, which caused structural changes in the modified molecules. The remaining fraction of formylated cytochrome c did not seem, however, to be affected by the presence of phospholipids (E, 7.0 = -0.085 V), but the experimental data were not accurate enough to draw definitive conclusions. Binding
Properties of Formylated chrome c
Cyto-
In contrast to native cytochrome c, its formylated derivative does not bind to the depleted mitochondria to an appreciable
extent. Figure 6 presents the binding of the two cytochromes c to c-depleted pigeon heart and rat liver mitochondria in the form of Scatchard plots. In order to look more carefully at the initial part of the binding curve, high concentrations of mitochondria were used. The results are expressed relative to cytochorme a concentration rather than per milligram of protein, since we consider the content of the former as a more reliable basis for the comparison of the experimental data. In aggrement with our previous results (11) Scatchard plots for the binding of native cytochrome c were nonlinear indicating heterogeneity in the binding sites. With native cytochrome c high affinity binding sites (with a K, of -0.02 pM) were present in the amount 2 per cytochrome a (estimated from extrapolation of the steeper part of the binding curve). Low affinity binding sites were present in considerably larger numbers and the binding constant was at least ten times higher. On the other hand, formylated cytochrome c binds only to a total of 2 per cytochrome a with a binding constant of -0.1 FM. K, of formylated cytochrome c for the nonspecific sites is much higher so that no nonspecific binding is observed.
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Reaction of Formylated Cytochrome c with Cytochrome c Oxidase and c Depleted Mitochondria The formylated cytochrome c (E,,,, = 0.085 V) was reduced by ascorbate in the absence and presence of TMPD and served as substrate for cytochrome c oxidase (Fig. 7) [the apparent K, of the reaction was dependent on TMPD concentration and extrapolated to a normal K, at infinite concentrations of TMPD-see also Margoliash et ai. (lo)]. Double reciprocal plots of the oxygen uptake against cytochrome c concentration (native or formylated) carried out at very low oxidase concentration yielded straight lines. From a comparison of the data it is seen that the apparent K, value of the oxidase for the formylated cytochrome c in sucrose/morpholinopropane sulfonate buffer of 1.2 PM is approximately six times higher than that for the native cytochrome c (0.2 PM). The apparent V with the formylated cytochrome c is, on the other hand, smaller. Essentially the same results were obtained with two different oxidase preparations, one isolated according to Kuboyama et al. (18), the other according to Sun et al. (19). On the other hand, in contrast to native cytochrome c, its formylated derivative did not stimulate the rate of oxygen uptake in the depleted mitochondria, in agreement with the findings of the other authors (5, 10). Moreover, when formylated cyto025
,s -5
-4
-3
I
-2
020 1
-I
0
f
I
234567
FIG. 7. Activities of native and formylated cytochrome c in the cytochrome c oxidase reaction. The reaction was carried out in 0.2 M sucrose-O.05 M morpholinopropane sulfonate pH 7.0 buffer at 24°C. 20 mM sodium ascorbate (pH 6.8) and 0.02 SM cytochrome c oxidase (preparation of Kuboyama et al., 1972, from pigeon breast). 1 mru TMPD was also present.
chrome c was added before the native molecule, the rate of oxygen uptake was smaller than in the absence of formylated derivative (Fig. 8). Increasing the concentration of formylated cytochrome c caused a progressive decrease of the respiratory rate at a constant concentration of native cytochrome c. The maximal respiratory rate that could be achieved was, however, the same in either the presence of or absence of formylated derivative, except that higher concentrations of native cytochrome c were required in the former case to obtain the maximum respiratory rate. This type of behavior is suggestive of unequal competition between the native and formylated cytochrome c for a common binding site and is confirmed by the Dixon plots of the experimental data which, as seen in Fig. 9, yield straight lines at two different cytochrome c concentrations. The apparent inhibitor constant calculated for formylated cytochrome c from this experimental data is 1.2 PM. Reaction of Formylated Cytochrome c with NADPH-C.ytochrome c Reductase Since the half-reduction potential of the formylated derivative of cytochrome c is nearly 0.2 V lower than that of the native c its reaction with cytochrome c, is thermodynamically not very favorable and may, in part, explain its lack of reactivity with mitochondrial NADH-cytochrome c reductase (with purified mitochondrial NADH-cytochrome c reductase, no reduction of exogenous cytochrome c was observed). In order to verify this suggestion we prepared microsomal NADPH-cytochrome c reductase which is known to donate the reducing equivalents at a much lower oxidation-reduction potential level. As seen in Table I, both the native cytochrome c and its formylated derivative were able to react with NADPH-cytochrome c reductase, although with different velocities. Native cytochrome c was reduced at a rate of 0.93 pmol/mg protein/ min, while the rate for formylated cytochrome c was only 0.19 pmollmg protein/ 20% of the rate min, i.e., approximately obtained with the native molecule. It
FORMYLATED
,r’ 05 025 [cytochrome~1 $4
FIG. 8. Restoration of oxygen uptake by cytochrome c in cytochrome c-depleted mitochondria in the presence (A) and absence (0) of formylated cytochrome c. Cytochrome c depleted rat liver mitochondria (0.7 mg protein/ml) were suspended in 0.2 M sucrose-O.050 M morpholinopropane sulfonate pH 7.0 buffer.
FIG. 9. Dixon plots of the inhibition of oxygen uptake by formylated cytochrome c. Rat liver mitochondria (0.7 mg protein/ml) were suspended in 0.2 M sucrosee0.050 M morpholinopropane sulfonate pH 7.0 buffer (0) 2 pM formyl c (A) 4 pM formyl c.
should, however, be pointed out that ferroformylated cytochrome c is autooxidizable and thus, the measured rates represent the steady-state rates which correspond to the differences between the rate of reduction of formylated cytochrome c by NADPH-cytochrome c reductase and the rate of its autooxidation by molecular oxygen. DISCUSSION
The experimental evidence (26,27) accumulated in the last few years suggests that the transfer of electrons in the mitochondrial respiratory chain occurs most likely through exchange between the molecules of similar oxidation-reduction potentials. The region of cytochrome c (E,,., = 0.235
CYTOCHROME
205
c
V) can be considered as one of the typical examples of such an electron exchange since cytochrome c reacts on one side with cytochrome c, [E,,., = 0.23 V (29, 30)] and on the other with cytochrome a [E,7.0 = 0.21 V (23)]. The measured steady-state levels of reduction of cytochromes c, c,, and a show that, in respiring mitochondria, they function at an E, value of 0.260-0.280 V (27). This means that under respiring conditions, formylated cytochrome c (Em,., = 0.085 V) would have to be less than 0.1% reduced. Since the rate of electron transfer between cytochrome c (or the formylated molecule) and the oxidase is proportional to the reduced form of the former, the observed velocity would be much smaller with formylated cytochrome. One can arrive at the same conclusion from yet another calculation. From the differences in the E, values between cytochromes c and c,, the value of the equilibrium constant between the two carriers can be calculated This equals 250 (-RT log K = -~FAE”‘). in the case of formylated cytochrome c, in contrast to the value of 1 for the native cytochromes in the mitochondria. If we further assume that the rate constant for the reduction of cytochrome c, by reduced formyl cytochrome c and by reduced cytochrome c are the same [k 1 = lo6 M- 1 s-l (2811, then the rate constant for the reduction of formyl cytochrome c by cytochrome c, must be less than that for native cytochrome c by 2.5 x lo*. TABLE
I
THE RATES OF REACTION BETWEEN THE MICROSOMAL NADPH-CYTOCHROME c REDWTASE AND CITOCHROMES co
Acceptor
Cytochrome Formylated
c cytochrome
Specific activity (fimollmg protein/min)
c
0.928 0.189
a The experimental conditions are given in Methods. The reductase activities were calculated from the initial rate of reduction of cytochrome c (or its formylated derivative). The millimolar extinction coefficient used for the calculation was 27.9 for cytochrome c and 21.3 for formylated cytochrome c. The reaction was carried out at 22°C.
206
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A single species of formylated cytochrome c (E,,,, = 0.085 V) used in this work was characterized by a half-reduction potential approximately 0.150 V more negative than that of its donor, cytochrome c,. Thus, even if the changed half-reduction potential were the only feature of the modification, one would expect on thermodynamic grounds that the formylated cytochrome c would not be very effective in restoring the oxygen uptake of the depleted mitochondria. Detailed thermodynamic and kinetic studies on formylated cytochrome c presented above demonstrate that formylation of tryptophan-59 induces structural changes in the cytochrome c molecule which are expressed in the change in its E, value and reactivity with the reductase and oxidase part of the respiratory chain. The spectral properties of formylated cytochrome c, the lack of the 695 nm band, its reactivity toward azide, cyanide, and carbon monoxide, and the appearance of a high spin heme signal below pH 6.5 indicate that methionine-80 bond is broken, in agreement with the finding of previous authors (5, 9). This may arise from the fact that the introduction of a formyl group on the indole ring nitrogen of tryptophan in between the sequence of aromatic residues located in the vicinity of heme (1, 2) causes changes in their mutual relationship. Moreover, the formyl residue is lypophilic and therefore its presence in the hydrophobic interior of cytochrome c may lead to a decrease in the stability of the protein because maximum entropy in the globular proteins (and therefore the most stable structure) is attained when their hydrophobic residues are buried inside while the hydrophylic amino acid residues are exposed to the surface (31, 32). The net positive charge on the surface of formylated cytochrome c should not change; the results show however that the affinity constant for the nonspecific binding sites increases by several orders of magnitude so that very little binding occurs. Perhaps the substitution of a formyl group for a hydrogen atom normally available for hydrogen bonding (see also 5) does affect the distribution of charges on the
surface of the molecule. Supporting evidence for this suggestion comes from the finding that the electrophoretic mobility of formylated cytochrome c is smaller than that of the native molecule (Asakura and Erecinska, unpublished data). Detailed investigations show that formylated cytochrome c binds to the high affinity, “specific” sites in cytochrome c-depleted mitochondria: first, formylated cytochrome c inhibits in a competitive manner the oxygen uptake induced by the addition of native cytochrome c to cytochrome c-depleted mitochondria; second, reduced formylated cytochrome c is readily oxidized by cytochrome c oxidase. Third, binding of formylated cytochrome c occurs at the precise stoichiometry of 2 cytochromes c per one cytochrome a, i.e., the same stoichiometry at which these two carriers are present in intact mitochondria. The same stoichiometry was obeyed when native cytochrome c is incorporated into the depleted membranes and the values are calculated by extrapolation from the initial steep part of the binding curve. An additional confirmation of this stoichiometry comes from the experiments on porphyrin cytochrome c which binds also with the ratio of 2 cytochromes c per one a at the high affinity binding sites (Erecinska and Vanderkooi, in preparation). The intimate nature of the specific binding sites remains to be elucidated although complexes of cytochrome c with cytochrome c oxidase of that stoichiometry have been isolated previously in vitro (33, 34, and for review see 35). This suggests that the isolated oxidase may carry on its surface some binding site(s) for cytochrome c; however, experimental evidence thus far available does not warrant the conclusion that cytochrome c is bound to cytochrome c oxidase in vitro. The formylated cytochrome c described in this work (Em,., = 0.085 V) was reducible by ascorbate in spite of the absence of the 695 nm band. The relationship between the intact 6th ligand and the reducibility by ascorbate is by no means clear. In some cases (36, 37), the disappearance of the 695 nm band occurs parallel with a decrease in reducibility by ascorbate, in others (38), the reactivity toward ascorbate remained
FORMYLATED
unaltered in spite of the fact that the 6th ligand’s position was occupied by an imidazole instead of a methionine. Since we did not measure the actual rates of reduction by ascorbate, it is possible that they were smaller than with the native cytochrome c; however, this neither interfered with our experiments nor affects the conelusion that formylated cytochrome c is readily oxidized by cytochrome c oxidase, in agreement with Margoliash et al. (10). The apparent K, value of the oxidase for formylated cytochrome c was approximately six times higher than for the native cytochrome c and the apparent V was slightly smaller. This decrease in the ap, parent Vmay suggest some modification of the electron transfer pathway. The undetectable activity of formylated cytochrome c in the reaction with mitochondrial NADH-cytochrome c reductase can be explained on the basis of a large unfavorable free energy change in the reduction of formylated cytochrome c (E, 7.0 = 0.085 V) by cytochrome c, (E, ,.. = 0.235 V). In line with this explanation is the tmding that formylated cytochrome c was able to accept the reducing equivalents from microsomal NADPH-cytochrome c reductase although the rates were only -20% of that with the unmodified molecule. And finally, a comment with respect to our second question posed in the introduction. The information presented in this work indicates that modification of a single amino acid residue, that of tryptophan-59, triggers important changes in the thermodynamic and catalytic properties of the molecule, most likely because of the position of tryptophan in the vicinity of heme. The differences in reactivity with cytochrome oxidase can be in part accounted for by the changes in binding properties of the modified molecule. Similarly, the lack of reactivity toward mitochondrial NADHcytochrome c reductase can be explained on thermodynamic grounds. However, the changes in the apparent V in the oxidase reaction and low activity with microsomal NADPH-cytochrome c reductase suggest that both the oxidation and the reduction pathways have been affected by formylation of the tryptophan residue. It should be
CYTOCHROME
207
c
stated finally that, although the experiments show that both pathways have been modified, they neither provide evidence for two pathways being identical nor do they exclude such a possibility. Moreover, they provide no information about the actual electron transfer pathways in and out of the cytochrome c molecule. ACKNOWLEDGMENTS Our thanks are due to Drs. H. R. Drott and R. Herschberg for their help in preparing the formylated cytochrome c and to Dr. D. F. Wilson for many stimulating discussions. The NMR spectra were obtained by Dr. J. Vanderkooi. REFERENCES 1. DICKERSON,R. E., TAKANO, T., EISENBERG,D., KALLAI, 0. B., SAMSON,L., COOPER,A., AND MARGOLIASH,E. (1971) J. Viol. Chem. 246, 1511-1535. 2. TAKANO, T., KALLAI, 0. B., SWANSON,R., AND DICKERSON,R. E. (1973) J. Biol. Chem. 248, 5234-5255. 3. WINFIELD,M. E. (1965) J. Mol. Biol. 12,600-611. 4. PREVIERO,A., COLEITI-PREVIERO,M. A., AND CAVADORE,J. C. (1967) Biochim. Biophys. Acta 147, 453-461. 5. AVIRAM, I., AND SCHEJTER,A. (1971) Biochim. Biophys. Acta 229,113-118. 6. ANDO, K., MATSUBARA,H., AND OKUNUKI, K. (1966) Biochim.
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AND JACOBS, E. E. (1968) Biochim. Biophys.
Acta 153, 804-818. 20. OMURA, T., AND TAKESUE, S. Biochem.(Japan) 67, 249-257.
(1970)
J.
21. JACOBS, E. E., AND SANADI, D. R. (1960) J. Viol. Chem. 235, 531-534. 22. BOVERIS, A., ERECI~SKA, M., AND WAGNER, M. (1972) Biochim. Biophys. Acta 256, 223-242. 23. WILSON, D. F., AND DU~ON, P. L. (1970) Arch. Biochem. Biophys. 136,583-584. 24. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M. (1949) J. Biol. Chem. 177, 751-766. 25. YONETANI, T. (1968) in Structure and Function of Cytochromes (Okunuki, K., Kamen, M. D., and Sekuzu, I., eds.), pp. 289-292, Univ. of Tokyo Press/&iv. Park Press, Tokyo and Baltimore. 26. WILSON, D. F., DUTTON, P. L., ERECI~SKA, M., LINDSAY, J. G., AND SATO, N. (1972) Accounts. Chem. Res. 5, 234-241. 27. WILSON, D. F., ERECI&SKA, M., AND DUWON, P. L. (1974) Ann. Reu. Biophys. Bioeng. 3, 203-230. 28. Yu, C. A., Yu, L., AND KING, T. E. (1973) J. Biol.
Chem. 248. 528-533.
29. GREEN, D. E., JXRNEFELT, J., AND TISDALE, H. D. (1959) Biochim. Biophyy. Acta 31, 34-46. 30. DUTTON, P. L., WILSON, D. F., AND LEE, C. P. (1970) Biochemistry 9, 5077-5082. 31. FRANK, H. W., AND EVANS, M. W. (1945) J. Chem. Phys. 13, 507-511. 32. TANFORD, C. (1962) J. Amer. Chem. Sot. 84,
4240-4247. 33. ORII, Y., SEKU+J, I., AND OKUNUKI, I. (1962) J. Biochem. (Tokyo) 51, 204-215. 34. KUBOYAMA, M., TAKEMORI, S., AND KING, T. E. (1962) Biochem. Biophys. Res. Commun. 9, 534-539. 35. NICHOLLS, P. (1974) Biochim. Biophys. Acta 346, 261-310.
36. GREENWOOD, C., AND PALMER, G. (1965) J. Biol. Chem. 240, 3660-3663. 37. GREENWOOD,C., AND WILSON, M. T. (1971 Eur. J. Biochem. 22, 5-10. 38. SKOV, K., AND WILLIAMS, G. R. (1968) in Structure and Function of Cytochromes (Okunuki, K., Kamen, M. D., and Sekuzu, I. eds.), pp. 349-352, Univ. of Tokyo Press/Univ. Park Press, Tokyo and Baltimore.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Cytochrome
169, 199-208 (I9751
c interaction
with
Formylated
Cytochrome
MARIA
ERECIfiSKA
University
of Pennsylvania,
Johnson Research Foundation,
Received March
Membranes c’
Philadelphia,
Pennsylvania
19174
1, 1975
A single species of tryptophan-59 formylated cytochrome c with a half-reduction potential of 0.085 * 0.01 V at pH 7.0 was used to study its catalytic and functional properties. The spectral properties of the modified cytochrome show that the 6th ligand position is open to reaction with azide, cyanide, and carbon monoxide. Formylated cytochrome c binds to cytochrome c depleted rat liver and pigeon heart mitochondria with the precise stoichiometry of two modified cytochrome c molecules per molecule of cytochrome a (K, of approx 0.1 PM). Formylated cytochrome c was reducible by ascorbate and was readily oxidized by cytochrome c oxidase. The apparent K, value of the oxidase for the formylated cytochrome c was six times higher than for the native cytochrome and the apparent V was smaller. Formylated cytochrome c does not restore the oxygen uptake in C-depleted mitochondria but inhibits, in a competitive manner, the oxygen uptake induced by the addition of native cytochrome c. Formylated cytochrome c was inactive in the reaction with mitochondrial NADH-cytochrome c reductase but was able to accept electrons through the microsomal NADPH-cytochrome c reductase.
Horse heart cytochrome c contains a single tryptophan residue at position 59 of its polypeptide chain. This tryptophan residue is situated in close proximity to the heme and to two other aromatic residues, those of tyrosines 67 and 74, in what appears on the X-ray structure as the left “hydrophobic channel” (1). The relative positions of the three aromatic residues to each other and to the heme are dependent on the redox state of cytochrome c. In the oxidized molecule (1) the plape of tryptophan 59 is approximately 5 A away from the heme plane and parallel to tyrosine 74. Upon reduction, tryptophan rotates against the heme so that the alignment between its ring and that of tyrosine 74 is broken and assumes a new position in which it becomes parallel to tyrosine 67 (2). Because of the ease with which the H electron systems of the aromatic amino acid residues share their electron “clouds,” ‘Fifth paper in the series. Papers I-IV 11-14. Supported by USPH GM 12202-10.
are Refs. 199
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
Winfield (3) put forward a suggestion that tyrosine and tryptophan radicals were involved in electron transfer through the protein network of hemeproteins. The existence of the left “hydrophobic channel” filled with aromatic residues and their particular influence upon the redox reactions of cytochrome c led Dickerson and co-workers (1, 2) to postulate that the “conduction mechanism” was responsible for the electron transfer through the cytochrome c molecule. Supporting evidence for such a mechanism of electron transfer may be provided by the studies on cytochrome c derivatives in which the abovementioned aromatic residues had been individually modified. The tacit assumptions are: 1. That such modification influences only the amino acid in question, and 2. That modification affects either the rate of electron donation or withdrawal or both. In 1967, Previero et al. (4) showed that tryptophan reacts quantitatively in a solution of formic acid and HCl to give only
MARIA
200
ERECIhSKA
one product in which the formyl group is linked to the nitrogen of the indole ring. Aviram and Schejter (5) used this procedure to formylate the tryptophan residue of cytochrome c. In the modified cytochrome, they noted in addition to the disappearance of the 280 nm absorbance, the absence of the 695 nm absorbance band in the oxidized molecule, characteristic of the intact methionine-80 bond (6-8). The absence of the upfield-shifted resonances corresponding to protons of the Fe-bound methionyl residue in formylated cytochrome c (9) was considered evidence for the confirmation of the results of Aviram and Schejter (5). The formylated cytochrome c was autooxidizable and bound CO; it could not be reduced by ascorbate and was inactive in restoring the respiration in cytochrome c-depleted mitochondria (5). On the other hand, Margoliash et al. (10) reported that formylated cytochrome c was equally as active as the native molecule in the reaction with cytochorme c oxidase, although it was inactive in the reaction with cytochrome c reductase. In continuation of our previous studies on cytochrome c interactions with membranes (ll-14), we decided to look more systematically at the reactions of formylated cytochrome c. The starting point of our investigation was the intriguing observation (10) that TMPD [E,,.,, = +260 mV and 30°C (15)], but not ascorbate [E,,,, = +45 mV (16)], was able to reduce formylated cytochrome c. The questions we asked were: 1. What are the thermodynamic and binding characteristics of formylated cytochrome c? 2. How much can be learned from studies on modified cytochromes c of the natural electron transfer pathways? This paper reports some information relevant to these two questions. A preliminary account of the work has already appeared (17). MATERIALS
AND
METHODS
Materials Formylated cytochrome c was prepared according to the method of Aviram and Schejter (5). After dialysis of the reacted cytochrome c against 10 mM phosphate buffer pH 7.2, as described in the original
method, the solution of diluted cytochrome c was adsorbed onto a small CM-cellulose column and the final concentrated formylated cytochrome c was eluted with 0.4 M phosphate buffer pH 7.4. Cytochrome c oxidase w-as isolated from pigeon breast mitochondria either by the method of Kuboyama et al. (18) or by the method of Sun et al. (19). Microsomal NADP-cytochrome c reductase was solubilized from rat liver microsomes by controlled digestion with trypsin as described by Omura and Takesue (20) and used without further purification. Rat liver cytochrome c-depleted mitochondria were prepared by the method of Jacobs and Sanadi (21), and pigeon heart depleted membranes by the modification of this method described by Boveris et al. (22). Methods Oxidation-reduction potential measurements. The potentiometric titrations of formylated cytochrome c were carried out by using simultaneous measurements of absorbance and oxidation-reduction potential (for details see Ref. 23). Potassium ferricyanide was used as an oxidant and freshly prepared sodium dithionite solution as a reductant. The redox mediators used were: diaminodurene (Aldrich Chem. Co., Milwaukee, WI), phenazine methosulfate (Sigma Chem. Co., St. Louis, MO), phenazine ethosulfate (K & K, Plainview, NY), duroquinone (Aldrich), 2-hydroxynaphthoquinone (K & K) and pyocyanine (K & K); their concentrations were lo-15 pM. Measurements were carried out at 22-25°C. Binding of cytochromes c was determined as the amount of native or formylated cytochrome c found in the mitochondrial pellet after centrifuging down the cytochrome c depleted mitochondria (10 min at SoOOg) from the suspending medium (0.20 M sucrose-0.05 M morpholinopropane sulfonate buffer pH 7.0). The pellet was suspended in 0.1 M phosphate buffer pH 7.2 containing 1% Triton X-100. The concentration of cytochrome c was estimated from the difference between total oxidized (+ 5 mM ferricyanide)-total reduced (t dithionite) measured at 550-540 nm. The extinction coefficients used were 19.7 for native cytochrome c and 13.4 for formylated cytochrome c. The concentration of free cytochrome c was determined by measuring the amount found in the supernatant from the 8000g centrifugation. Oxygen uptake was measured at 24°C with a Clark oxygen electrode in 0.20 M sucrose-O.050 M morpholinopropane sulfonate pH 7.2 buffer, in the presence of succinate plus glutamate as substrate. Cytochrome oxidase activity was determined by measuring the rates of oxygen uptake at 24°C with a Clark oxygen electrode. Parallel experiments were carried out for the native and formylated cytochrome c. The results were corrected for the autoxidation rates of cytochromes c. The detailed conditions are given in the figure legends.
FORMYLATED NADPH-cytochrome c reductase activity was measured spectrophotometrically by following the rate of cytochrome c reduction (or formylated cytochrome c) at 550 nm. The assay medium contained: 2 x 10m5M cytochrome c, 1 x lo-’ M NADPH, and enzyme in 0.1 M phosphate buffer, pH 7.2. Total volume was 3 ml. The reaction was started by the addition of NADPH. Cyanide was omitted from the reaction mixture since it reacted with formylated ferricytochrome c. It was found that the rates of enzymatic oxidation of ferrocytochrome c were negligible, because partially purified NADPH-cytochrome c reductase was used for assays. Cytochrome a concentration was measured at 605-630 nm (reduced-oxidized) using an extinction coefficient of 24. Protein was determined by the biuret method (24). RESULTS
Spectral Characteristics of Formylated Cytochrome c
CYTOCHROME
201
c
28 Ii I’
24
I i
,20 16
E .I 2 -8
520
540 560
580
X (nm)
FIG. 1. Light absorption spectrum of horse heart cytochrome c (broken line) and its formylated derivative (solid line). Cytochrome c (or formylated cytochrome c) was suspended in 0.1 M phosphate buffer pH 7.0 at a concentration of 15-20 fiM.
The spectral characteristics of oxidized formylated cytochrome c have been described by Aviram and Schejter (5) and can be summarized as follows: 1. Disappearance of the 280 nm absorbance peak and simultaneous increase in absorbance at around 298 nm; 2. 3-4 nm shift in the Soret maximum toward shorter wavelength; 3. disappearance of the 695 nm 360 420 460 500520 560 6ocl 640 absorbance band. In addition to the Wovelengfh , nmI Wwelengfh I “In I changes in the spectrum of the oxidized FIG. 2. Absorption spectra of formylated cytomolecule, we have noted that the extincchrome c and its cyano and azide compounds. Oxition coefficients for the reduced absorption dized formylated cytochrome c was suspended in 0.1 bands of formylated cytochrome c differ M phosphate buffer pH 7.0 at a concentration of 16.5 from those for the native molecule: the pM (-) formylated cytochrome c; (-----) 15 mM extinction coefficient at 550 nm for the sodium azide; (- -. ~. ) 15 mM potassium cyanide. reduced cytochrome decreases from 27.9 (native cytochrome c) to 21.3 (formylated mylated cytochrome c has its (Y maximum cytochrome c). The calculations based on at 536 nm and Soret maximum at 415 nm; the data of Fig. 1 yield a value of 13.4 for the azide formylated cytochrome c peaks reduced minus oxidized extinction coeffiare at 537 and 412 for the (Y and Soret cient at 550 minus 540 nm for formylated maximums, respectively (the formylated cytochrome c. The Soret maximum of the cytochrome c exhibited peaks at 528 and reduced cytochrome is shifted 2-3 nm t.o- 405 nm). The reduced form of formylated ward the red and the extinction coefficient cytochrome c reacts with carbon monoxide is increased by -10% upon formylation. It as manifested by the decrease in absorpshould be pointed out that similar changes tion in the (Y region and an increase in the in the cytochrome c molecule were ob- Soret region. The Soret maximum of the CO compound of formylated ferrocytoserved by Yonetani (25) when it was modichrome c is at 416 nm. fied by N-bromosuccinamide addition. The spectrum of oxidized formylated Oxidized formylated cytochrome c reacts cytochrome c is pH dependent (Fig. 3). readily at neutral pH with added ligands, Lowering the pH causes the appearance of cyanide, and azide (Fig. 2). The cyanofor-
202
MARIA
ERECIfVSKA
(data not shown) and was essentially identical of the data of Wfithrich et al. (9). Thermodynamic
500
550
600
650
.Ahd FIG. 3. Effect of pH on absorption spectra of formylated cytochrome c. The buffers used were mixtures of 0.1 M citric acid and 0.2 M K,HPO, of appropriate pH. 17.8 FM formylated cytochrome c.
0:I
I Ox/Red
I'0
FIG. 4. Potentiometric titration curve of formylated cytochrome c. Formylated cytochrome c was suspended at a concentration of 5 pM in 0.2 M sucrose-O.050 M morpholinopropane sulfonate pH 7.0 buffer. The redox mediators used were: 20 pM diaminodurene, 40 pM each of phenazine methosulfate, phenazine ethosulfate and duroquinone, 5 FM pyocyanine, and 15 PM 2-OH, 1,4 naphtoquinone (0) oxidative titration with potassium ferricyanide; (A) reductive titration with sodium dithionite.
an absorption peak at 618-620 nm, characteristic of high spin compounds. Below pH 6.5 formylated cytochrome c is a mixture of high and low spin forms. The latter information is confirmed by EPR spectroscopy which shows a gradual increase in the peak with a g value of 6.0 when the pH is lowered (not shown). The NMR spectrum of oxidized formylated cytochrome c showed the absence of the upfield shifted resonances at 3.7, 3.3, and 2.7 ppm corresponding to the protons of the iron-bound methionyl-80 residue
Properties of Formylated Cytochrome c
Anaerobic potentiometric titrations of formylated cytochrome c prepared as described by Aviram and Schejter (5) yield a sigmoid titration curve, characteristic of two components (Fig. 4). Resolution of this titration curve into the two components gives the values of their half-reduction potentials: 0.090 f 0.01 V for the higher potential one and -0.055 V for the lower. Both components titrate with a slope of 1 which indicates that they are one-electron donors/acceptors. When formylated cytochrome c, purified on a small CM-cellulose column is titrated under the same conditions only one component is observed with a half-reduction potential of 0.085 V (i.e., it corresponds to the high potential component of the nonpurified formylated cytochrome c) (Fig. 5). All of the studies described below were done with formylated cytochrome c with an E,,.O of 0.085 V. Treatment of formylated cytochrome c at alkaline pH (5) causes displacement of the formyl group and return of the spectral properties to those of the native form. The thermodynamic properties of formylated cytochrome c also revert to their original value; the midpoint potential of .
FIG. 5. Potentiometric titration of CM-cellulose purified formylated and deformylated cytochrome c. Deformylation was carried out by alkaline treatment as described by Aviram and Schejter (1969) 5.1 FM formylated cytochrome c, 4.8 PM deformylated cytochrome c. Conditions are those of Fig. 5.
FORMYLATED
CYTOCHROME
c
203
FIG. 6. Binding of native and formylated cytochromes c to cytochrome c-depleted rat liver and pigeon heart mitochondria. Cytochrome c depleted mitochondria (at least 1 pM cytochrome a) were incubated in 0.20 M sucrose-O.050 M morpholinopropane sulfonate pH 7.0 buffer with various amounts of cytochromes c for 5 min at room temperature. The buffer was presaturated with oxygen and the samples aerated during the incubation time. The mitochondria were removed by centrifugation at 8OOOg for 10 min at 4°C. The pellet was suspended in 0.1 M phosphate buffer pH 7.2 containing 1% triton X-100. The concentration of cytochromes c in the pellet and in the supernate was determined as described in Methods; (0, 0) horse heart cytochrome C; (A, A) formylated horse heart cytochrome c.
“deformylated” cytochrome c was found to be indistinguishable from that for the native molecule (Fig. 5). Attempts to measure the half-reduction potential of the formylated cytochrome c in the presence of negatively charged phospholipid vesicles were rather unsuccessful. The molecule appeared to be labile-a fraction of the 0.085 V component was converted into a form which was very difficult to reduce which suggested to us possible denaturation of the formylated cytochrome c. Since a relatively high concentration of cardiolipin was used in these experiments, the local density of negative charges may have been very high, which caused structural changes in the modified molecules. The remaining fraction of formylated cytochrome c did not seem, however, to be affected by the presence of phospholipids (E, 7.0 = -0.085 V), but the experimental data were not accurate enough to draw definitive conclusions. Binding
Properties of Formylated chrome c
Cyto-
In contrast to native cytochrome c, its formylated derivative does not bind to the depleted mitochondria to an appreciable
extent. Figure 6 presents the binding of the two cytochromes c to c-depleted pigeon heart and rat liver mitochondria in the form of Scatchard plots. In order to look more carefully at the initial part of the binding curve, high concentrations of mitochondria were used. The results are expressed relative to cytochorme a concentration rather than per milligram of protein, since we consider the content of the former as a more reliable basis for the comparison of the experimental data. In aggrement with our previous results (11) Scatchard plots for the binding of native cytochrome c were nonlinear indicating heterogeneity in the binding sites. With native cytochrome c high affinity binding sites (with a K, of -0.02 pM) were present in the amount 2 per cytochrome a (estimated from extrapolation of the steeper part of the binding curve). Low affinity binding sites were present in considerably larger numbers and the binding constant was at least ten times higher. On the other hand, formylated cytochrome c binds only to a total of 2 per cytochrome a with a binding constant of -0.1 FM. K, of formylated cytochrome c for the nonspecific sites is much higher so that no nonspecific binding is observed.
204
MARIA
ERECIkSKA
Reaction of Formylated Cytochrome c with Cytochrome c Oxidase and c Depleted Mitochondria The formylated cytochrome c (E,,,, = 0.085 V) was reduced by ascorbate in the absence and presence of TMPD and served as substrate for cytochrome c oxidase (Fig. 7) [the apparent K, of the reaction was dependent on TMPD concentration and extrapolated to a normal K, at infinite concentrations of TMPD-see also Margoliash et ai. (lo)]. Double reciprocal plots of the oxygen uptake against cytochrome c concentration (native or formylated) carried out at very low oxidase concentration yielded straight lines. From a comparison of the data it is seen that the apparent K, value of the oxidase for the formylated cytochrome c in sucrose/morpholinopropane sulfonate buffer of 1.2 PM is approximately six times higher than that for the native cytochrome c (0.2 PM). The apparent V with the formylated cytochrome c is, on the other hand, smaller. Essentially the same results were obtained with two different oxidase preparations, one isolated according to Kuboyama et al. (18), the other according to Sun et al. (19). On the other hand, in contrast to native cytochrome c, its formylated derivative did not stimulate the rate of oxygen uptake in the depleted mitochondria, in agreement with the findings of the other authors (5, 10). Moreover, when formylated cyto025
,s -5
-4
-3
I
-2
020 1
-I
0
f
I
234567
FIG. 7. Activities of native and formylated cytochrome c in the cytochrome c oxidase reaction. The reaction was carried out in 0.2 M sucrose-O.05 M morpholinopropane sulfonate pH 7.0 buffer at 24°C. 20 mM sodium ascorbate (pH 6.8) and 0.02 SM cytochrome c oxidase (preparation of Kuboyama et al., 1972, from pigeon breast). 1 mru TMPD was also present.
chrome c was added before the native molecule, the rate of oxygen uptake was smaller than in the absence of formylated derivative (Fig. 8). Increasing the concentration of formylated cytochrome c caused a progressive decrease of the respiratory rate at a constant concentration of native cytochrome c. The maximal respiratory rate that could be achieved was, however, the same in either the presence of or absence of formylated derivative, except that higher concentrations of native cytochrome c were required in the former case to obtain the maximum respiratory rate. This type of behavior is suggestive of unequal competition between the native and formylated cytochrome c for a common binding site and is confirmed by the Dixon plots of the experimental data which, as seen in Fig. 9, yield straight lines at two different cytochrome c concentrations. The apparent inhibitor constant calculated for formylated cytochrome c from this experimental data is 1.2 PM. Reaction of Formylated Cytochrome c with NADPH-C.ytochrome c Reductase Since the half-reduction potential of the formylated derivative of cytochrome c is nearly 0.2 V lower than that of the native c its reaction with cytochrome c, is thermodynamically not very favorable and may, in part, explain its lack of reactivity with mitochondrial NADH-cytochrome c reductase (with purified mitochondrial NADH-cytochrome c reductase, no reduction of exogenous cytochrome c was observed). In order to verify this suggestion we prepared microsomal NADPH-cytochrome c reductase which is known to donate the reducing equivalents at a much lower oxidation-reduction potential level. As seen in Table I, both the native cytochrome c and its formylated derivative were able to react with NADPH-cytochrome c reductase, although with different velocities. Native cytochrome c was reduced at a rate of 0.93 pmol/mg protein/ min, while the rate for formylated cytochrome c was only 0.19 pmollmg protein/ 20% of the rate min, i.e., approximately obtained with the native molecule. It
FORMYLATED
,r’ 05 025 [cytochrome~1 $4
FIG. 8. Restoration of oxygen uptake by cytochrome c in cytochrome c-depleted mitochondria in the presence (A) and absence (0) of formylated cytochrome c. Cytochrome c depleted rat liver mitochondria (0.7 mg protein/ml) were suspended in 0.2 M sucrose-O.050 M morpholinopropane sulfonate pH 7.0 buffer.
FIG. 9. Dixon plots of the inhibition of oxygen uptake by formylated cytochrome c. Rat liver mitochondria (0.7 mg protein/ml) were suspended in 0.2 M sucrosee0.050 M morpholinopropane sulfonate pH 7.0 buffer (0) 2 pM formyl c (A) 4 pM formyl c.
should, however, be pointed out that ferroformylated cytochrome c is autooxidizable and thus, the measured rates represent the steady-state rates which correspond to the differences between the rate of reduction of formylated cytochrome c by NADPH-cytochrome c reductase and the rate of its autooxidation by molecular oxygen. DISCUSSION
The experimental evidence (26,27) accumulated in the last few years suggests that the transfer of electrons in the mitochondrial respiratory chain occurs most likely through exchange between the molecules of similar oxidation-reduction potentials. The region of cytochrome c (E,,., = 0.235
CYTOCHROME
205
c
V) can be considered as one of the typical examples of such an electron exchange since cytochrome c reacts on one side with cytochrome c, [E,,., = 0.23 V (29, 30)] and on the other with cytochrome a [E,7.0 = 0.21 V (23)]. The measured steady-state levels of reduction of cytochromes c, c,, and a show that, in respiring mitochondria, they function at an E, value of 0.260-0.280 V (27). This means that under respiring conditions, formylated cytochrome c (Em,., = 0.085 V) would have to be less than 0.1% reduced. Since the rate of electron transfer between cytochrome c (or the formylated molecule) and the oxidase is proportional to the reduced form of the former, the observed velocity would be much smaller with formylated cytochrome. One can arrive at the same conclusion from yet another calculation. From the differences in the E, values between cytochromes c and c,, the value of the equilibrium constant between the two carriers can be calculated This equals 250 (-RT log K = -~FAE”‘). in the case of formylated cytochrome c, in contrast to the value of 1 for the native cytochromes in the mitochondria. If we further assume that the rate constant for the reduction of cytochrome c, by reduced formyl cytochrome c and by reduced cytochrome c are the same [k 1 = lo6 M- 1 s-l (2811, then the rate constant for the reduction of formyl cytochrome c by cytochrome c, must be less than that for native cytochrome c by 2.5 x lo*. TABLE
I
THE RATES OF REACTION BETWEEN THE MICROSOMAL NADPH-CYTOCHROME c REDWTASE AND CITOCHROMES co
Acceptor
Cytochrome Formylated
c cytochrome
Specific activity (fimollmg protein/min)
c
0.928 0.189
a The experimental conditions are given in Methods. The reductase activities were calculated from the initial rate of reduction of cytochrome c (or its formylated derivative). The millimolar extinction coefficient used for the calculation was 27.9 for cytochrome c and 21.3 for formylated cytochrome c. The reaction was carried out at 22°C.
206
MARIA
ERECIhSKA
A single species of formylated cytochrome c (E,,,, = 0.085 V) used in this work was characterized by a half-reduction potential approximately 0.150 V more negative than that of its donor, cytochrome c,. Thus, even if the changed half-reduction potential were the only feature of the modification, one would expect on thermodynamic grounds that the formylated cytochrome c would not be very effective in restoring the oxygen uptake of the depleted mitochondria. Detailed thermodynamic and kinetic studies on formylated cytochrome c presented above demonstrate that formylation of tryptophan-59 induces structural changes in the cytochrome c molecule which are expressed in the change in its E, value and reactivity with the reductase and oxidase part of the respiratory chain. The spectral properties of formylated cytochrome c, the lack of the 695 nm band, its reactivity toward azide, cyanide, and carbon monoxide, and the appearance of a high spin heme signal below pH 6.5 indicate that methionine-80 bond is broken, in agreement with the finding of previous authors (5, 9). This may arise from the fact that the introduction of a formyl group on the indole ring nitrogen of tryptophan in between the sequence of aromatic residues located in the vicinity of heme (1, 2) causes changes in their mutual relationship. Moreover, the formyl residue is lypophilic and therefore its presence in the hydrophobic interior of cytochrome c may lead to a decrease in the stability of the protein because maximum entropy in the globular proteins (and therefore the most stable structure) is attained when their hydrophobic residues are buried inside while the hydrophylic amino acid residues are exposed to the surface (31, 32). The net positive charge on the surface of formylated cytochrome c should not change; the results show however that the affinity constant for the nonspecific binding sites increases by several orders of magnitude so that very little binding occurs. Perhaps the substitution of a formyl group for a hydrogen atom normally available for hydrogen bonding (see also 5) does affect the distribution of charges on the
surface of the molecule. Supporting evidence for this suggestion comes from the finding that the electrophoretic mobility of formylated cytochrome c is smaller than that of the native molecule (Asakura and Erecinska, unpublished data). Detailed investigations show that formylated cytochrome c binds to the high affinity, “specific” sites in cytochrome c-depleted mitochondria: first, formylated cytochrome c inhibits in a competitive manner the oxygen uptake induced by the addition of native cytochrome c to cytochrome c-depleted mitochondria; second, reduced formylated cytochrome c is readily oxidized by cytochrome c oxidase. Third, binding of formylated cytochrome c occurs at the precise stoichiometry of 2 cytochromes c per one cytochrome a, i.e., the same stoichiometry at which these two carriers are present in intact mitochondria. The same stoichiometry was obeyed when native cytochrome c is incorporated into the depleted membranes and the values are calculated by extrapolation from the initial steep part of the binding curve. An additional confirmation of this stoichiometry comes from the experiments on porphyrin cytochrome c which binds also with the ratio of 2 cytochromes c per one a at the high affinity binding sites (Erecinska and Vanderkooi, in preparation). The intimate nature of the specific binding sites remains to be elucidated although complexes of cytochrome c with cytochrome c oxidase of that stoichiometry have been isolated previously in vitro (33, 34, and for review see 35). This suggests that the isolated oxidase may carry on its surface some binding site(s) for cytochrome c; however, experimental evidence thus far available does not warrant the conclusion that cytochrome c is bound to cytochrome c oxidase in vitro. The formylated cytochrome c described in this work (Em,., = 0.085 V) was reducible by ascorbate in spite of the absence of the 695 nm band. The relationship between the intact 6th ligand and the reducibility by ascorbate is by no means clear. In some cases (36, 37), the disappearance of the 695 nm band occurs parallel with a decrease in reducibility by ascorbate, in others (38), the reactivity toward ascorbate remained
FORMYLATED
unaltered in spite of the fact that the 6th ligand’s position was occupied by an imidazole instead of a methionine. Since we did not measure the actual rates of reduction by ascorbate, it is possible that they were smaller than with the native cytochrome c; however, this neither interfered with our experiments nor affects the conelusion that formylated cytochrome c is readily oxidized by cytochrome c oxidase, in agreement with Margoliash et al. (10). The apparent K, value of the oxidase for formylated cytochrome c was approximately six times higher than for the native cytochrome c and the apparent V was slightly smaller. This decrease in the ap, parent Vmay suggest some modification of the electron transfer pathway. The undetectable activity of formylated cytochrome c in the reaction with mitochondrial NADH-cytochrome c reductase can be explained on the basis of a large unfavorable free energy change in the reduction of formylated cytochrome c (E, 7.0 = 0.085 V) by cytochrome c, (E, ,.. = 0.235 V). In line with this explanation is the tmding that formylated cytochrome c was able to accept the reducing equivalents from microsomal NADPH-cytochrome c reductase although the rates were only -20% of that with the unmodified molecule. And finally, a comment with respect to our second question posed in the introduction. The information presented in this work indicates that modification of a single amino acid residue, that of tryptophan-59, triggers important changes in the thermodynamic and catalytic properties of the molecule, most likely because of the position of tryptophan in the vicinity of heme. The differences in reactivity with cytochrome oxidase can be in part accounted for by the changes in binding properties of the modified molecule. Similarly, the lack of reactivity toward mitochondrial NADHcytochrome c reductase can be explained on thermodynamic grounds. However, the changes in the apparent V in the oxidase reaction and low activity with microsomal NADPH-cytochrome c reductase suggest that both the oxidation and the reduction pathways have been affected by formylation of the tryptophan residue. It should be
CYTOCHROME
207
c
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