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Effect of pH on the electrochemical and spectrophotometric properties of Pseudomonas aeruginosa cytochrome c551: A comparison with horse heart cytochrome c
Bioelectrochemistry and Bioenergetics, 11 (1983) 319-326 A section of J. Electroanal. Chem., and constituting Vol. 156 (1983) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
319
590--EFFECT OF pH ON THE ELECTROCHEMICAL AND SPECTROPHOTOMETRIC PROPERTIES OF PSEUDOMONA S AERUGINOSA CYTOCHROME Cssl: A COMPARISON WITH HORSE HEART CYTOCHROME c
JEAN HALADJIAN and PIERRE BIANCO
Laboratoire de Chimie et Electrochimie des Complexes, Universitb de Provence, Place Victor Hugo, 13331 Marseille Cedex 3 (France) (Revised manuscript received October 15th 1983)
SUMMARY Cytochrome c551 from Pseudomonas aeruginosa has been studied by differential pulse voltammetry and cyclic voltammetry at the 4,4'-bipyridine activated gold electrode within the pH range 4.3-10.9. The redox potential U~ varies from ca. 0.32 to 0.26 V (versus n.h.e.). Spectrophotometric measurements show the disappearance of the 690 nm band, an enhancement of the 408 nm band, and the appearance of an additional band at 551 nm when the pH is increased from 6 to 11, but without total reversibility when the p H is returned to the initial value. The existence of two forms of cytochrome c551 (iron-methionine bonded and iron-methionine unbonded heme) is proposed, and a comparison with horse heart cytochrome c is given.
INTRODUCTION
The bacterial cytochrome c551 from Pseudomonas aeruginosa [1] can be considered an ancestor of mitochondrial cytochrome c [2,3]. For instance, in the case of horse heart cytochrome c and P. aeruginosa cytochrome c551, the single heme group is bound through two thioether linkages to the polypeptide chain, and both have histidine and methionine as the fifth and sixth iron ligands. However, cytochrome c55l has only 82 amino acids and an acid isoelectric point (4.7) [1] in contrast to horse heart cytochrome c, which has 104 amino acids and an alkaline isoelectric point (10.4) [2]. The electrochemical properties of the heme group in c-type cytochromes seem to be controlled by two important factors: the nature of the iron ligands and the immediate proteic environment. One way of disturbing the heme environment and of modifying the iron ligands is to vary the pH. This investigation can be performed by monitoring redox properties, especially the redox potential [4] and the electrochemical reversibility at a working electrode as a function of pH. Differential pulse voltammetry (d.p.v.) and cyclic voltammetry (c.v.) at the 4,4'-bipyridiiae activated 0302-4598/83/$03.00
© 1983 Elsevier Sequoia S.A.
320 gold electrode are useful tools for studying horse heart cytochrome c at p H 7 [5-7]. Recent papers have shown that this technique can also be applied to a more extended p H range [8] and to other c-type cytochromes [9,10]. Perturbations of the heme environment of c-type cytochromes can also be demonstrated by modifications of their visible absorption spectra; effectively, the integrity of the iron-methionine bond is directly related to the presence of the 695 n m band [2]. Thus the p H dependence of the electroactivity of cytochrome c [8] and cytochrome c551 was studied by using voltammetry in conjunction with spectrophotometry. EXPERIMENTAL
Materials Cytochrome c55~ from P. aeruginosa was a kind gift of Dr. M. Bruschi, Laboratoire de Chimie Bact6rienne, Marseille. It was prepared and purified as described previously [1]. Horse heart cytochrome c (type VI, from Sigma Chemical Co.) was used without further purification. All other chemicals were reagent grade.
Methods and apparatus The working electrode used for voltammetric measurements was the 4,4'-bipyridine activated gold electrode [5-7]. All other experimental details have been given in previous papers [8,9]. RESULTS
Cyclic voltammetry In the absence of 4,4'-bipyridine, no voltammetric signal was detected with the gold electrode. Cyclic voltammograms for 0.015 M potassium phosphate medium, p H 7.2, containing 0.01 M 4,4'-bipyridine and 143 /~M cytochrome c [1] or cytochrome c551 [2] are shown in Fig. l a for a scan rate, v, of 2 mV s -1. For cytochrome c, well-developed cathodic and anodic peaks separated by about 60 mV were observed (curve 1); this is significant of a fast electrochemical system [5]. For cytochrome c551, one cathodic and one anodic peak were detected in curve 2 but their heights were lower and the separation between the reduction and reoxidation peaks, AUe, reached about 100 mV for the scan rate used. The peak currents varied linearly with v ~/2, as expected for a diffusion-controlled process. Thus it appears that cytochrome c551 acts as a less rapid monoelectronic system than cytochrome c at the 4,4'-bipyridine activated gold electrode. From the scan-rate dependence of AUp, it was possible to calculate the heterogeneous rate constant for the electron transfer between cytochrome c551 and the activated gold electrode using the Nicholson treatment [11]; a value of 3.2 × 1 0 - 4 c m S-1 was obtained for k S. This value is
321
< ! / ]0 ....
U(Vr's n h e y
.....
, /
"#'S
W" -10
/
c
I
-20
/,'~
t
I
\ %
%
-° ~
•
.oO°o•, I
-0,2
I
....... ; ....... ( ( V q 0.2
°o oo
n.h.e.! 0.4
Fig. 1. Cyclic voltammograms (a) and d.p. voltammograms (b) at the 4A'-bipyridine activated gold electrode of 143 ffM horse heart cytochrome c (1) and P. aeruginosa cytochrome c55l (2) in (0.015 M potassium phosphate +0.01 M 4,4'-bipyridine) buffer solution, pH 7.2. Scan rate v = 2 mV s - l ; pulse amplitude AU = - 2 5 mV; pulse repetition rate = 2 s 1. ( ..... ) Background solution.
noticeably lower than the value of approximately 1.6 × 10 2 cm s- 1, which has been previously determined for horse heart cytochrome c [6]. Nevertheless, cytochrome c551 may be regarded as a quasi-reversible electrochemical system [12]. Thus from the midpoint between the forward and reverse peak potentials, the redox potential, U~, of the system can be estimated with good approximation. A value of 0.26 V v e r s u s n.h.e, was determined at pH 7.2, and is in
322 agreement with the data obtained by Moore et al. [4] from spectrophotometric and potentiometric measurements. The effect of pH on cyclic voltammograms of cytochrome c55~ was investigated over the p H range 4.3-10.9 in 0.015 M potassium phosphate +0.015 M sodium borate + 0.01 M 4,4'-bipyridine medium, after adjustments with sodium hydroxide or acetic acid. The c.v. peaks were well-shaped only between pH 4.8 and 9.4. The pH dependence of the redox potential of cytochrome c551 calculated from the c.v. curves is shown in Fig. 2 (curve 1) and is in good agreement with the data of Moore et al. [4]. The values obtained by Margalit and Schejter [13] for cytochrome c within the same pH range are given in Fig. 2 (curve 2) for comparison.
Differential pulse voltammetry Cytochrome c55] was studied by d.p.v, using the 4,4'-bipyridine activated gold electrode; d.p. voltammograms obtained at pH 7.2 for the same 143/~M concentration are given in Fig. l b for comparison with cytochrome c. As noted above for the c.v. peaks, the d.p.v, reduction peak was noticeably lower with cytochrome c551 than with cytochrome c. This decrease could be due to a less complete reversibility of the electrochemical system. The effect of pH on d.p. voltammograms of cytochrome cs5] is shown in Fig. 3. The d.p.v, peak observed at pH 7.2 was absent at pH 4.3 and above pH 10.1; its height increased from pH 4.8 to 6.7, then decreased between pH 6.7 and 10.1. The peak potential Up became more and more positive when the pH decreased below 7.2. It remained unchanged above pH - 7.2, as previously noted for the variation of U~. Moreover, it was observed that the d.p.v, peak was fully restored when the pH of the cytochrome Csst solution was lowered to 4.3 and then raised back to 7.2. But when the pH was increased from 7.2 to 10.9 and then lowered back to 7.2, the d.p.v, peak was not totally restored; moreover, an additional slight peak was detected at Up - 0.05 V versus n.h.e. (Fig. 3, dashed curve).
;-0.%, 0,300 ~'~
2
"~,~ 0.100
I
I
I
I
5
6
7
8
pH
I
I
I
9
10
11
Fig. 2. pH dependence of the redox potential of P. aeruginosa cytochrome cs51 (1) and horse heart cytochrome c (2) (data from Margalit and Schejter [13]). U~ values for cytochromecss1 were calculated from c.v. results.
323
9.4
i'i
U (Vvs n.h.e,) -0.1
I
I
I
i
i
0
0.1
0.2
0.3
0.4
Fig. 3. Effect of pH on the d.p. voltammograms at the activated gold electrode of 143 t~M P. aeruginosa cytochrome c551 in 0.015 M potassium phosphate +0.015 M sodium borate +0.01 M 4,4'-bipyridine medium (same experimental conditions as in Fig. lb). (-- -- --) pt-I 7.2 after adjustment at pH 10.9, then returned to initial pit. Spectrophotometry In agreement with previous papers [14,15], we noted that the absorption spectrum of ferricytochrome c551 remained virtually unchanged within the p H range 7.2-9.5. W h e n the p H increased above this value, the 690 n m b a n d was lowered and then disappeared at p H - 11 (Fig. 4). Simultaneously, an enhancement of the 408 n m band, accompanied by a slight b a t h o c h r o m i c effect, and the appearance above p H - 1 0 of the 551 n m b a n d were observed; these optical modifications are usually detected when ferrocytochrome c551 is formed. Thus the alkalisation of the cytoc h r o m e c551 solution appears to be accompanied by the formation of the reduced form. W h e n the p H was lowered to the original value the absorption spectra of
324 1,5
403 nm
E:
1.3
/ 1.4 0
I
1,1
/
8
I
pH
I
I
/
J
10
0
?
-1.2
690 nrn
o/
0,1
8
I0
0,06
pH I I I I 8 10 Fig. 4. pH dependence ot the optical absorption of P. aeruginosa cytochrome c551at 408, 551 and 690 nm. Increasing (O) then decreasing pH (@). The arrows indicate the direction in which the pH was varied (i.e. forward and ~ backward). 0.02
I
6
ferricytochrome c551 obtained previously were not fully restored: the 690 n m b a n d only partly reappeared (Fig. 4), the bands associated with the reduced form did not disappear, and an additional b a n d appeared at 580 nm. C o n t r a r y to the case of horse heart cytochrome c [8] and Desulfovibrio vulgaris cytochrome c553 [9], the transition from the neutral to the alkaline form of P. aeruginosa cytochrome c551 between p H 9.5 and 11 is not reversible; consequently, we were unable to calculate the transition p K in the t h e r m o d y n a m i c sense. Moreover, it is probable that a new species is irreversibly formed at alkaline pH; this result is consistent with the foregoing d.p.v, results (Fig. 3, dashed curve). DISCUSSION In the p H range 4.8-7.5, the redox potential of P. aeruginosa is p H - d e p e n d e n t (Fig. 2), in agreement with the results of Moore et al. [4]. This p H dependence can be due to the existence of two ionizations affecting the redox potential, one in the oxidized form and the other in the reduced form [4], or more schematically to the existence of an equilibrium between two cytochrome c55~ forms, a neutral one with a redox potential U~ of 0.26 versus n.h.e., which predominates at 7.5 _< p H _< 9.4, and
325 an acid one, which begins to appear when the pH decreases below 7.5, with a likely more positive but not determined redox potential U~ of >_ 0.30 V versus n.h.e. The nature of the ionizing group, i.e. hemic propionic acid side chains, has been cleared up from n.m.r, measurements by Chao et al. [14] and Moore et al. [4]. By comparing cytochrome c and cytochrome c551, it seems likely that the propionic acids of cytochrome c551 are more accessible and less hydrogen-bonded than in mitochondrial cytochrome ¢; thus the properties of the heme in cytochrome c55~ may be perturbed by these ionizations, contrarily to the case of cytochrome c. The shift observed for U~ from 0.30 to 0.26 V could correspond to a slight increase in the percentage exposure of the heine to the solvent [16]; this interpretation agrees with the disruption of some hydrogen bonds allowing better accessibility of the heme. When the pH is raised to 10.9, d.p.v, and spectrophotometric measurements show that cytochrome c551 is transformed into a new alkaline form, but this transformation is not reversible, as in the case of cytochrome c; the absorption spectrum of the native protein is not entirely restored (Fig. 4), an additional d.p.v, peak and an absorption band (at ~ = 580 nm) appearing even when the pH returns to its initial value of 7.2. The disappearance of the 690 nm band is probably due to the disruption of the iron-methionine bond which occurs at alkaline pH (10-11). For cytochrome c, it is well known that the conversion of methionine 80- to lysine-bonded heme results from deprotonation of the next lysine residue (probably lysine 79). A similar conversion may exist for D. vulgaris cytochrome c553, as the methionine (probably methionine 64), which is linked to the hemic iron, should be very close to two lysine residues [17]. In the case of P. aeruginosa cytochrome c551, no lysine residue is present in the immediate vicinity of its methionine 61-iron ligand; thus it is likely that no other lysine residue is able to occupy the sixth position freed through disruption of the methionine 61-iron bond. It has been suggested [14] that the sixth ligand could be the hydroxide, O H - . Anyway, it seems that the proteic structure of this iron-methionine unbonded form of cytochrome c55~ becomes more flimsy after alkalisation, resulting in the appearance of reduced cytochrome c55~. The mechanism of this reduction when the pH is increased has not been elucidated so far, but we can assume that it is accompanied by simultaneous oxidation of part of the cyrochrome c55~; the appearance of the 580 nm absorption band should be attributed to this undetermined oxidized form. It is interesting to note that 4,4'-bipyridine has an activating effect on cytochrome c551 [10], as in the case of horse heart cytochrome c and D. vulgaris cytochrome c553, but the electrochemical system denotes a less-marked reversibility. Moreover, 4,4'-bipyridine becomes inefficient under pH values of approximately 4.8 and above pH values of approximately 9.4. This may be due to the appearance of new forms of cytochrome c551 in acid and alkaline solutions. It has been suggested that the activating power of 4,4'-bipyridine may be due to hydrogen-bonding of the protein lysine residues to the adsorbed 4,4'-bipyridine nitrogen lone-pairs [6]. From the comparison between the amino acid composition of both proteins, it appears that horse heart cytochrome c contains a higher lysine percentage than cytochrome c55~
326 TABLE 1 Comparison between some properties of horse heart cytochrome c and P. aeruginosa cytochrome c551
No. of amino acids No. of lysine residues Isoelectric point U~(V) (n.h.e.) at pH 7.0 p K (met.-to lys.-bonded heine) Heterogeneous rate constant k s (at the 4,4'-bipyridine activated gold electrode) (cm s 1)
h.h. cyt. c
P. aerug,
104 19 10.4 0.26 9.1 a
82 8 4.7 0.26 Not determined
1.4-1.9 × 10 - 2 b
cyt. c551
3.2 × 10 - 4 c
a Data from ref. [2]. b Data from Eddowes et al. [6]. c Present paper.
(Table 1), and this could be the reason for the less-pronounced activating power of 4,4'-bipyridine in the presence of cytochrome cs5~. ACKNOWLEDGEMENT
We thank Dr. M. Bruschi for the gift of a sample of purified protein and for helpful discussions on this paper. REFERENCES 1 R.P. Ambler, Biochem. J., 89 (1963) 341. 2 R.E. Dickerson and R. Timkovich in The Enzymes, Vol. 11, P.D. Boyer (Editor), Academic Press, New York, 1975, pp. 397-547. 3 R. Dickerson, Pour la Science, 31 (1980) 60. 4 G.R. Moore, G.W. Pettigrew, R.C. Pitt and R.J.P. Williams, Biochim. Biophys. Acta, 590 (1980) 261. 5 M.J. Eddowes and H.A.O. Hill, J. Am. Chem. Soc., 101 (1979) 4461. 6 M.J. Eddowes, H.A.O. Hill and K. Uosaki, Bioelectrochem. Bioenerg., 7 (1980) 527. 7 W.J. Albery, M.J. Eddowes, H.A.O. Hill and A.R. Hillman, J. Am. Chem. Soc., 103 (1981) 3904. 8 J. Haladjian, R. Pilard, P. Bianco and P.A. Serre, Bioelectrochem. Bioenerg., 9 (1982) 91. 9 P. Bianco, J. Haladjian, R. Pilard and M. Bruschi, J. Electroanal. Chem., 136 (1982) 291. 10 P. Bianco, J. Haladjian, M. Loutfi and M. Bruschi, Biochem. Biophys. Res. Commun., 113 (1983) 526. 11 R.S. Nicholson, Anal. Chem., 37 (1965) 1351. 12 A.M. Bond, Modern Polarographic Methods in Analytical Chemistry, Marcel Dekker, New York and Basel, 1980, p. 24. 13 R. Margalit and A. Schejter, Eur. J. Biochem., 32 (1973) 492. 14 Y.H. Chao, R. Bersohn and P. Aisen, Biochemistry, 18 (1979) 774. 15 S.N. Vinogradov, Biopolymers, 9 (1970) 507. 16 E. Stellwagen, Nature (London), 275 (1978) 73. 17 M. Bruschi and J. Le Gall, Biochim. Biophys. Acta, 271 (1972) 48.
319
590--EFFECT OF pH ON THE ELECTROCHEMICAL AND SPECTROPHOTOMETRIC PROPERTIES OF PSEUDOMONA S AERUGINOSA CYTOCHROME Cssl: A COMPARISON WITH HORSE HEART CYTOCHROME c
JEAN HALADJIAN and PIERRE BIANCO
Laboratoire de Chimie et Electrochimie des Complexes, Universitb de Provence, Place Victor Hugo, 13331 Marseille Cedex 3 (France) (Revised manuscript received October 15th 1983)
SUMMARY Cytochrome c551 from Pseudomonas aeruginosa has been studied by differential pulse voltammetry and cyclic voltammetry at the 4,4'-bipyridine activated gold electrode within the pH range 4.3-10.9. The redox potential U~ varies from ca. 0.32 to 0.26 V (versus n.h.e.). Spectrophotometric measurements show the disappearance of the 690 nm band, an enhancement of the 408 nm band, and the appearance of an additional band at 551 nm when the pH is increased from 6 to 11, but without total reversibility when the p H is returned to the initial value. The existence of two forms of cytochrome c551 (iron-methionine bonded and iron-methionine unbonded heme) is proposed, and a comparison with horse heart cytochrome c is given.
INTRODUCTION
The bacterial cytochrome c551 from Pseudomonas aeruginosa [1] can be considered an ancestor of mitochondrial cytochrome c [2,3]. For instance, in the case of horse heart cytochrome c and P. aeruginosa cytochrome c551, the single heme group is bound through two thioether linkages to the polypeptide chain, and both have histidine and methionine as the fifth and sixth iron ligands. However, cytochrome c55l has only 82 amino acids and an acid isoelectric point (4.7) [1] in contrast to horse heart cytochrome c, which has 104 amino acids and an alkaline isoelectric point (10.4) [2]. The electrochemical properties of the heme group in c-type cytochromes seem to be controlled by two important factors: the nature of the iron ligands and the immediate proteic environment. One way of disturbing the heme environment and of modifying the iron ligands is to vary the pH. This investigation can be performed by monitoring redox properties, especially the redox potential [4] and the electrochemical reversibility at a working electrode as a function of pH. Differential pulse voltammetry (d.p.v.) and cyclic voltammetry (c.v.) at the 4,4'-bipyridiiae activated 0302-4598/83/$03.00
© 1983 Elsevier Sequoia S.A.
320 gold electrode are useful tools for studying horse heart cytochrome c at p H 7 [5-7]. Recent papers have shown that this technique can also be applied to a more extended p H range [8] and to other c-type cytochromes [9,10]. Perturbations of the heme environment of c-type cytochromes can also be demonstrated by modifications of their visible absorption spectra; effectively, the integrity of the iron-methionine bond is directly related to the presence of the 695 n m band [2]. Thus the p H dependence of the electroactivity of cytochrome c [8] and cytochrome c551 was studied by using voltammetry in conjunction with spectrophotometry. EXPERIMENTAL
Materials Cytochrome c55~ from P. aeruginosa was a kind gift of Dr. M. Bruschi, Laboratoire de Chimie Bact6rienne, Marseille. It was prepared and purified as described previously [1]. Horse heart cytochrome c (type VI, from Sigma Chemical Co.) was used without further purification. All other chemicals were reagent grade.
Methods and apparatus The working electrode used for voltammetric measurements was the 4,4'-bipyridine activated gold electrode [5-7]. All other experimental details have been given in previous papers [8,9]. RESULTS
Cyclic voltammetry In the absence of 4,4'-bipyridine, no voltammetric signal was detected with the gold electrode. Cyclic voltammograms for 0.015 M potassium phosphate medium, p H 7.2, containing 0.01 M 4,4'-bipyridine and 143 /~M cytochrome c [1] or cytochrome c551 [2] are shown in Fig. l a for a scan rate, v, of 2 mV s -1. For cytochrome c, well-developed cathodic and anodic peaks separated by about 60 mV were observed (curve 1); this is significant of a fast electrochemical system [5]. For cytochrome c551, one cathodic and one anodic peak were detected in curve 2 but their heights were lower and the separation between the reduction and reoxidation peaks, AUe, reached about 100 mV for the scan rate used. The peak currents varied linearly with v ~/2, as expected for a diffusion-controlled process. Thus it appears that cytochrome c551 acts as a less rapid monoelectronic system than cytochrome c at the 4,4'-bipyridine activated gold electrode. From the scan-rate dependence of AUp, it was possible to calculate the heterogeneous rate constant for the electron transfer between cytochrome c551 and the activated gold electrode using the Nicholson treatment [11]; a value of 3.2 × 1 0 - 4 c m S-1 was obtained for k S. This value is
321
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U(Vr's n h e y
.....
, /
"#'S
W" -10
/
c
I
-20
/,'~
t
I
\ %
%
-° ~
•
.oO°o•, I
-0,2
I
....... ; ....... ( ( V q 0.2
°o oo
n.h.e.! 0.4
Fig. 1. Cyclic voltammograms (a) and d.p. voltammograms (b) at the 4A'-bipyridine activated gold electrode of 143 ffM horse heart cytochrome c (1) and P. aeruginosa cytochrome c55l (2) in (0.015 M potassium phosphate +0.01 M 4,4'-bipyridine) buffer solution, pH 7.2. Scan rate v = 2 mV s - l ; pulse amplitude AU = - 2 5 mV; pulse repetition rate = 2 s 1. ( ..... ) Background solution.
noticeably lower than the value of approximately 1.6 × 10 2 cm s- 1, which has been previously determined for horse heart cytochrome c [6]. Nevertheless, cytochrome c551 may be regarded as a quasi-reversible electrochemical system [12]. Thus from the midpoint between the forward and reverse peak potentials, the redox potential, U~, of the system can be estimated with good approximation. A value of 0.26 V v e r s u s n.h.e, was determined at pH 7.2, and is in
322 agreement with the data obtained by Moore et al. [4] from spectrophotometric and potentiometric measurements. The effect of pH on cyclic voltammograms of cytochrome c55~ was investigated over the p H range 4.3-10.9 in 0.015 M potassium phosphate +0.015 M sodium borate + 0.01 M 4,4'-bipyridine medium, after adjustments with sodium hydroxide or acetic acid. The c.v. peaks were well-shaped only between pH 4.8 and 9.4. The pH dependence of the redox potential of cytochrome c551 calculated from the c.v. curves is shown in Fig. 2 (curve 1) and is in good agreement with the data of Moore et al. [4]. The values obtained by Margalit and Schejter [13] for cytochrome c within the same pH range are given in Fig. 2 (curve 2) for comparison.
Differential pulse voltammetry Cytochrome c55] was studied by d.p.v, using the 4,4'-bipyridine activated gold electrode; d.p. voltammograms obtained at pH 7.2 for the same 143/~M concentration are given in Fig. l b for comparison with cytochrome c. As noted above for the c.v. peaks, the d.p.v, reduction peak was noticeably lower with cytochrome c551 than with cytochrome c. This decrease could be due to a less complete reversibility of the electrochemical system. The effect of pH on d.p. voltammograms of cytochrome cs5] is shown in Fig. 3. The d.p.v, peak observed at pH 7.2 was absent at pH 4.3 and above pH 10.1; its height increased from pH 4.8 to 6.7, then decreased between pH 6.7 and 10.1. The peak potential Up became more and more positive when the pH decreased below 7.2. It remained unchanged above pH - 7.2, as previously noted for the variation of U~. Moreover, it was observed that the d.p.v, peak was fully restored when the pH of the cytochrome Csst solution was lowered to 4.3 and then raised back to 7.2. But when the pH was increased from 7.2 to 10.9 and then lowered back to 7.2, the d.p.v, peak was not totally restored; moreover, an additional slight peak was detected at Up - 0.05 V versus n.h.e. (Fig. 3, dashed curve).
;-0.%, 0,300 ~'~
2
"~,~ 0.100
I
I
I
I
5
6
7
8
pH
I
I
I
9
10
11
Fig. 2. pH dependence of the redox potential of P. aeruginosa cytochrome cs51 (1) and horse heart cytochrome c (2) (data from Margalit and Schejter [13]). U~ values for cytochromecss1 were calculated from c.v. results.
323
9.4
i'i
U (Vvs n.h.e,) -0.1
I
I
I
i
i
0
0.1
0.2
0.3
0.4
Fig. 3. Effect of pH on the d.p. voltammograms at the activated gold electrode of 143 t~M P. aeruginosa cytochrome c551 in 0.015 M potassium phosphate +0.015 M sodium borate +0.01 M 4,4'-bipyridine medium (same experimental conditions as in Fig. lb). (-- -- --) pt-I 7.2 after adjustment at pH 10.9, then returned to initial pit. Spectrophotometry In agreement with previous papers [14,15], we noted that the absorption spectrum of ferricytochrome c551 remained virtually unchanged within the p H range 7.2-9.5. W h e n the p H increased above this value, the 690 n m b a n d was lowered and then disappeared at p H - 11 (Fig. 4). Simultaneously, an enhancement of the 408 n m band, accompanied by a slight b a t h o c h r o m i c effect, and the appearance above p H - 1 0 of the 551 n m b a n d were observed; these optical modifications are usually detected when ferrocytochrome c551 is formed. Thus the alkalisation of the cytoc h r o m e c551 solution appears to be accompanied by the formation of the reduced form. W h e n the p H was lowered to the original value the absorption spectra of
324 1,5
403 nm
E:
1.3
/ 1.4 0
I
1,1
/
8
I
pH
I
I
/
J
10
0
?
-1.2
690 nrn
o/
0,1
8
I0
0,06
pH I I I I 8 10 Fig. 4. pH dependence ot the optical absorption of P. aeruginosa cytochrome c551at 408, 551 and 690 nm. Increasing (O) then decreasing pH (@). The arrows indicate the direction in which the pH was varied (i.e. forward and ~ backward). 0.02
I
6
ferricytochrome c551 obtained previously were not fully restored: the 690 n m b a n d only partly reappeared (Fig. 4), the bands associated with the reduced form did not disappear, and an additional b a n d appeared at 580 nm. C o n t r a r y to the case of horse heart cytochrome c [8] and Desulfovibrio vulgaris cytochrome c553 [9], the transition from the neutral to the alkaline form of P. aeruginosa cytochrome c551 between p H 9.5 and 11 is not reversible; consequently, we were unable to calculate the transition p K in the t h e r m o d y n a m i c sense. Moreover, it is probable that a new species is irreversibly formed at alkaline pH; this result is consistent with the foregoing d.p.v, results (Fig. 3, dashed curve). DISCUSSION In the p H range 4.8-7.5, the redox potential of P. aeruginosa is p H - d e p e n d e n t (Fig. 2), in agreement with the results of Moore et al. [4]. This p H dependence can be due to the existence of two ionizations affecting the redox potential, one in the oxidized form and the other in the reduced form [4], or more schematically to the existence of an equilibrium between two cytochrome c55~ forms, a neutral one with a redox potential U~ of 0.26 versus n.h.e., which predominates at 7.5 _< p H _< 9.4, and
325 an acid one, which begins to appear when the pH decreases below 7.5, with a likely more positive but not determined redox potential U~ of >_ 0.30 V versus n.h.e. The nature of the ionizing group, i.e. hemic propionic acid side chains, has been cleared up from n.m.r, measurements by Chao et al. [14] and Moore et al. [4]. By comparing cytochrome c and cytochrome c551, it seems likely that the propionic acids of cytochrome c551 are more accessible and less hydrogen-bonded than in mitochondrial cytochrome ¢; thus the properties of the heme in cytochrome c55~ may be perturbed by these ionizations, contrarily to the case of cytochrome c. The shift observed for U~ from 0.30 to 0.26 V could correspond to a slight increase in the percentage exposure of the heine to the solvent [16]; this interpretation agrees with the disruption of some hydrogen bonds allowing better accessibility of the heme. When the pH is raised to 10.9, d.p.v, and spectrophotometric measurements show that cytochrome c551 is transformed into a new alkaline form, but this transformation is not reversible, as in the case of cytochrome c; the absorption spectrum of the native protein is not entirely restored (Fig. 4), an additional d.p.v, peak and an absorption band (at ~ = 580 nm) appearing even when the pH returns to its initial value of 7.2. The disappearance of the 690 nm band is probably due to the disruption of the iron-methionine bond which occurs at alkaline pH (10-11). For cytochrome c, it is well known that the conversion of methionine 80- to lysine-bonded heme results from deprotonation of the next lysine residue (probably lysine 79). A similar conversion may exist for D. vulgaris cytochrome c553, as the methionine (probably methionine 64), which is linked to the hemic iron, should be very close to two lysine residues [17]. In the case of P. aeruginosa cytochrome c551, no lysine residue is present in the immediate vicinity of its methionine 61-iron ligand; thus it is likely that no other lysine residue is able to occupy the sixth position freed through disruption of the methionine 61-iron bond. It has been suggested [14] that the sixth ligand could be the hydroxide, O H - . Anyway, it seems that the proteic structure of this iron-methionine unbonded form of cytochrome c55~ becomes more flimsy after alkalisation, resulting in the appearance of reduced cytochrome c55~. The mechanism of this reduction when the pH is increased has not been elucidated so far, but we can assume that it is accompanied by simultaneous oxidation of part of the cyrochrome c55~; the appearance of the 580 nm absorption band should be attributed to this undetermined oxidized form. It is interesting to note that 4,4'-bipyridine has an activating effect on cytochrome c551 [10], as in the case of horse heart cytochrome c and D. vulgaris cytochrome c553, but the electrochemical system denotes a less-marked reversibility. Moreover, 4,4'-bipyridine becomes inefficient under pH values of approximately 4.8 and above pH values of approximately 9.4. This may be due to the appearance of new forms of cytochrome c551 in acid and alkaline solutions. It has been suggested that the activating power of 4,4'-bipyridine may be due to hydrogen-bonding of the protein lysine residues to the adsorbed 4,4'-bipyridine nitrogen lone-pairs [6]. From the comparison between the amino acid composition of both proteins, it appears that horse heart cytochrome c contains a higher lysine percentage than cytochrome c55~
326 TABLE 1 Comparison between some properties of horse heart cytochrome c and P. aeruginosa cytochrome c551
No. of amino acids No. of lysine residues Isoelectric point U~(V) (n.h.e.) at pH 7.0 p K (met.-to lys.-bonded heine) Heterogeneous rate constant k s (at the 4,4'-bipyridine activated gold electrode) (cm s 1)
h.h. cyt. c
P. aerug,
104 19 10.4 0.26 9.1 a
82 8 4.7 0.26 Not determined
1.4-1.9 × 10 - 2 b
cyt. c551
3.2 × 10 - 4 c
a Data from ref. [2]. b Data from Eddowes et al. [6]. c Present paper.
(Table 1), and this could be the reason for the less-pronounced activating power of 4,4'-bipyridine in the presence of cytochrome cs5~. ACKNOWLEDGEMENT
We thank Dr. M. Bruschi for the gift of a sample of purified protein and for helpful discussions on this paper. REFERENCES 1 R.P. Ambler, Biochem. J., 89 (1963) 341. 2 R.E. Dickerson and R. Timkovich in The Enzymes, Vol. 11, P.D. Boyer (Editor), Academic Press, New York, 1975, pp. 397-547. 3 R. Dickerson, Pour la Science, 31 (1980) 60. 4 G.R. Moore, G.W. Pettigrew, R.C. Pitt and R.J.P. Williams, Biochim. Biophys. Acta, 590 (1980) 261. 5 M.J. Eddowes and H.A.O. Hill, J. Am. Chem. Soc., 101 (1979) 4461. 6 M.J. Eddowes, H.A.O. Hill and K. Uosaki, Bioelectrochem. Bioenerg., 7 (1980) 527. 7 W.J. Albery, M.J. Eddowes, H.A.O. Hill and A.R. Hillman, J. Am. Chem. Soc., 103 (1981) 3904. 8 J. Haladjian, R. Pilard, P. Bianco and P.A. Serre, Bioelectrochem. Bioenerg., 9 (1982) 91. 9 P. Bianco, J. Haladjian, R. Pilard and M. Bruschi, J. Electroanal. Chem., 136 (1982) 291. 10 P. Bianco, J. Haladjian, M. Loutfi and M. Bruschi, Biochem. Biophys. Res. Commun., 113 (1983) 526. 11 R.S. Nicholson, Anal. Chem., 37 (1965) 1351. 12 A.M. Bond, Modern Polarographic Methods in Analytical Chemistry, Marcel Dekker, New York and Basel, 1980, p. 24. 13 R. Margalit and A. Schejter, Eur. J. Biochem., 32 (1973) 492. 14 Y.H. Chao, R. Bersohn and P. Aisen, Biochemistry, 18 (1979) 774. 15 S.N. Vinogradov, Biopolymers, 9 (1970) 507. 16 E. Stellwagen, Nature (London), 275 (1978) 73. 17 M. Bruschi and J. Le Gall, Biochim. Biophys. Acta, 271 (1972) 48.