4726
J. Phys. Chem. 1994,98, 4726-4728
pH and Temperature Effect on the Absorption Spectra of Pseudomonas aeruginosa Cytochrome c-551 Solution Yongfang Li,*J***s Kenichi Imaeda,* and Hiroo Inokuchit Institute for Molecular Science, Myodaiji, Okazaki 444, Japan, and Toyota Physical and Chemical Research Institute, Nagakute, Aichi 480- 11, Japan Received: August 17, 1993; In Final Form: February 14, 1994'
The temperature dependence of the visible-near-infrared absorption spectra of Pseudomonas aeruginosa cytochrome c-551 aqueous solutions was studied in the temperature range 2&80 OC. For a neutral or acidic solution the absorbance at 695 nm decreases and that at 620 nm increases with increasing temperature, which possibly corresponds to the transition from the low-spin state to the high-spin state of the ferricytochrome c-55 1 heme a t a higher temperature. For an alkaline solution the absorbances a t 551 and 521 nm, which are the characteristic absorptions of ferrocytochrome c-55 1, increased with incresing p H value of the solution and with increasing temperature. I t was speculated that the spectral change in alkaline solution resulted from the cleavage of the chemical bond between the heme iron and methionine-61 sulfur then probably followed by combination of OH- with the heme iron.
Cytochrome c-55 1 from Pseudomonas aeruginosa is a small C-type cytochrome of the bacterial respiration chain, by which an electron can be transferred from aruzin to cytochrome cd2.lJ This electorn transfer system in Ps. aeruginosa is regarded as a good analogue of that in mitochondria. Cytochrome c-551 resembles cytochrome c in tertiary structure. Both cytochrome c and c-55 1 have a single heme group covalently b u n d through two thioether linkages to cysteines of the amino acid chain and have a histidine and a methionine as the fifth and sixth iron ligands, re~pectively.~ The main difference is a large amino acid deletion at the bottom of cytochrome ~ - 5 5 1 Cytochrome .~ c-551 has 82 amino acid residues and an acidic isoelectric point (PI= 4.7)5 in contrast to horse heart cytochrome c, which has 104 amin acid residues and an alkaline isoelectric point (PI = 10.4).' The absorption spectra of cytochrome c solution have been studied d e e ~ l y . The ~ , ~ 695-nm band, which is intimately related to the overall conformation of the protein moiety of cytochrome c, was abolished by increasing the temperature and was found to be very sensitive to solution pH v a l ~ e s . ~The J visible absorption spectrum of cytochrome c-551 solution is similar to that of cytochrome c a t room temperature. Chao et al.9 studied the pH effect on the structure of cytochrome 0551. In the studies of the electron transfer properties of cytochrome c-551, the authors found that a 3-year aged cytochrome c-551 sample possesses a novel phase transition phenomenon in the temperature range 40-60 O C , I O while a newly prepared c-551 does not have this property. The conductivity of the aged c-55 1 solid film increased by ca. 2 orders after the phase transition at higher temperature. So, if we can make the structural change of the aged sample clear, it will be helpful to understand the electron tranfer mechanism in cytochrome c-551. Chao et al. suggested that some irreversibly denatured cytochrome 0 5 5 1 species exist in the 3-year aged sample.9 Usually, protein is easily denatured at higher temperatures or at extreme p H conditions. Therefore, in order to look for the origin of the phase transition phenomenon of the 3-year aged cytochrome c-55 1 sample and to checkthestability and thedenature behaviorofcytochromec-551, the pH and temperature dependence of the absorption spectra of cytochrome c-551 solutions was studied in this paper. t Permanent address: Institute of Chemistry, Academia Sinica, Beijing 100080, China. t Institute for Molecular Science. 1 Toyota Physical and Chemical Research Institute. CI Abstract published in Advance ACS Abstracts, March 15, 1994.
Experimental Section
Ps. aeruginosa cytochrome 0551 was purchased from the Sigma Co. (Lot NO. 70H4027). The visible and near-infrared absorption spectra of a 0.1 15 mM cytochrome c-551 aqueous solution were measured by a Hitachi U-3400 spectrophotometer. The temperature of the solution was controlled in the range 2085 OC with a temperature changeable water-cycling apparatus. The pH values of the sample solutions were regulated by 1 N HCl or 1 N KOH solution and were measured with a Horiba F-1 1 pH meter.
Results and Discussion pH Effect. Figure 1 shows the pH dependence of the visible absorption spectra of ferricytochrome c-551 solutions a t 20 OC. In the acidic solution (pH 7), the 695-nm peak also decreases gradually with increasing pH value, and it disappears at ca. pH 10. At the same time the absorption peaks at 551 and 521 nm, which are the characteristic bands of the reduced form of cytochrome c-55 1, appear and become stronger and stronger with increasing p H value. This phenomenon was not observed in ferricytochrome c solution. the 695-nm peak originated from the heme iron combined with methionine sulfur at its sixth ligand position.l.12 The disappearance of the 695-nm band for higher pH solutions (pH > 10) indicates that the bond between the heme iron and methionine sulfur breaks. Chao et al. suggested from
0022-365419412098-4126%04.50/0 0 1994 American Chemical Society
pH/Temperature Effects on Cytochrome c-55 1 Spectra
The Journal of Physical Chemistry, Vol. 98, No. 17, 1994 4727
Q+ 0
I -
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600
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4 /*
8002 o'
Wavelengthfn m,
Figure 1. pH effect on the visible absorption spectra of ferricytochrome 0551 solution at 20 OC.
1..
b
a
Figure 3. (a) Bonding structure of ferricytochrome c-551 heme iron in
the solution at pH 4.5. (b) Speculated bonding structure of ferricytochrome c-551 heme iron in the solution at pH >lo.
0 ' 3 tA 1.o,
A
-
$R1 u ' L [
\;
n
0 0.0 600
800
1000
1200
Wavelengt h/nm
Figure 2. Near-infrared absorption spectra of ferricytochrome c-551 solutions at (1) pH 1.57, (2) pH 2.07, and (3) pH 3.03 at 20 OC.
a variety of optical and ESR spectral changes that the methionine61 sulfur is replaced by other ligands at pH >9.4.9 The appearance of the absorption peaks of its reduced form implies that the cytochrome c-55 1 heme accepted some electrons in the alkaline solutions. Since OH- is a very strong nucelophilic reagent. One possibility is that the OH- ion replaces methionine-61 as the sixth ligand of the heme iron in the alkaline solutions. Then the negative charge in the OH- ion transfers to Fe(II1) partially, resulting in the appearance of the 551- and 521-nm absorption bands. The speculated structure is shown in Figure 3b. Temperature Effect. The temperature dependence of the absorption spectra of the ferricytochrome c-55 1 solutions of pH 7. In an acidic solution, the 695-nm peak decreases and the 620-nm peak increases with increasing temperature, as shown in Figure4. The spectralchange with increasing temperature is just like that with a decreasing pH value. Figure 4 also compares the temperature dependence of the visible absorption spectra of ferricytochrome c-55 1 solutions at pH 1.57, pH 2.07, and pH 3.03. It can be seen that the absorption spectrum of the pH 2.07 solution at 40 OC is similar to that of the pH 1.57 solution at 20 OC, and the absorption spectrum of the pH 3.03 solution at 50 O C is similar to that of the pH 2.07 solution at 20 OC. In other words, the absorption spectrum of a ferricytochrome c-551 acidic solution a t a higher temperature is similar to that of its lower pH solution at 20 O C . Since the 620-nm band is related to the transition from the lowspin state, to the high-spin state, as mentioned above, the increment of the 620-nm peak intensity implies that ferricytochrome 0551 in the acidic solutions turns to the high-spin state with increasing temperature.
Figure 4. Temperature effect of the visible absorption spectra of
ferricytochromec-551 solutions at pH 1.57
(-a),
pH 2.07 (- - -), and pH
3.03 (-).
n
Wavelength/n
m
Figure 5. Temperature effect of the visible absorption spectra of ferricytochrome c-551 solution at pH 9.50.
The absorption spectra of the ferricytochrome c-55 1 solution at pH 3.30 in the range 600-1200 nm were also measured from room temperature to60 OC. At 60 O C theabsorption bandaround lOOOnmappears, with theincrementofthe620-nm peakintensity, which is similar to that of the pH 1.57 solution a t 20 OC (see Figure 2). Figure 5 shows the temperature dependence of the visible absorption spectra of ferricytochrome c-551 solution at pH 9.7. It can be seen that the 695-nm peak is very weak at room temperature. When the temperature is increased from 20 to 60 OC, the 695-nm peak disappears gradually while the absorption peaks a t 551 and 521 nm become stronger and stronger. This behavior is similar to the spectra change with increasing pH value a t 20 "C in the alkaline solutions. It is reasonable to think that
4728 The Journal of Physical Chemistry, Vol. 98, No. 17, 1994
620nmband
4 1
20
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Li et al. was investigated in the temperature range 20-80 OC at different pH values. The spectral changes with temperature strongly depend on the pH values of the solutions. For a neutral or acidic solution, the absorbance at 695 nm decreases and that 620 nm increases with increasing temperature. Accordingly, it was suggested that the transition from the low-spin state to the highspin state of ferricytochrome c-551 takes place at higher temperatures. For an alkaline solution, 551- and 521-nm bands, which are thecharacteristic absorptionsof ferrocytochromec-551, appear and become stronger and stronger with increasing pH value and with increasing temperature. A pH-temperature phase diagram for the 695- and 620-nm bands is presented for a neutral or acidic solution of ferricytochrome c-551.
Temperature/'C
Figure 6. pH-temperature phase diagram of the 695- and 620-nm bands in the absorption spectra of ferricytochrome c-551 solutions at p H
