2832
Journal of the American Chemical Society
/ 102:s /
April 9, 1980
Kinetics and Mechanism of Reduction of Horse Heart Cytochrome c by Hexaammineruthenium( 11) Ion. Reactivities of the Electronic Isomers of Cytochrome c Adeleye Adegite* and M. I. Okpanachi Contribution from the Departments of Chemistry and Biochemistry, Ahmadu Bello Unicersity, Zaria, Nigeria. Receiced March 26, 1979
Abstract: The rate constant for the reduction of cytochrome c by R u ( N H ~ )shows ~ ~ + a complex dependence on p H . At pH 6 2 in a chloride medium, the rate constant at 25 “C, I = 0.1 M NaCI, is (35O/[H+]) M-l s-l, while at pH 24.0 the rate constant is (3.9 X IO4 1.0 X IO*[H+]) M-’ s-’. A maximum redox reactivity exists around pH 3, and in this region, neither of these
+
limiting expressions describes the dependence of the rate constant on pH. In perchlorate medium, the rate constant for the region [H+] = 0.02-0.1 M is (1.9 X IO4 + 4.6 X 1OZ[H+])M-’ s-l. The electron transfer from the reductant to the ferric atom in the protein is supposed to proceed by the heme edge mechanism. The results in the different media suggest that the reactivity order for the reduction of the acid-induced “electronic” isomers of cytochrome c by the heme edge mechanism is low spin > mixed spin > high spin. It is proposed that in vivo electron transfer may be preceded by a conformational change such that cytochrome c adopts the most reactive form, which contains one more redox-linked ionizable proton than the native conformer.
Introduction Cytochrome c is a relatively small metalloprotein (mol wt I2 384) that acts as an electron carrier in the respiratory chain of all aerobic organisms.’ X-ray ~ t u d i e s have ~ - ~ revealed that the heme group is located in a crevice of the essentially globular protein. The iron atom lies in the plane of the porphyrin ring and the fifth and sixth coordination positions are respectively occupied by a nitrogen atom of the imidazole ring of His-1 8 and the sulfur atom of Met-80. I n aqueous solutions, the coordination environment of the heme iron depends on the pH, the ionic strength, and the anionic composition of the medi ~ m .At~ physiological ? ~ pH, the coordination environment of the iron is believed to be the same as in the solid. However, at low pH the Fe-N and Fe-S bonds are both broken, and these coordination positions are probably occupied by water.’ At pH high spin > mixed spin.” It was also speculated that the relative rates for the mixed- and high-spin species may be inverted in electron transfer by the remote attack mechanism.” Ewall and Bennettih have reported their findings on the reduction of cytochrome c by R u ( N H ~ ) ~Their ~ + . results do show that electron transfer from this ion to cytochrome c proceeds by the remote attack mechanism. However, their studies were conducted in the range 3.3 < pH 9 7.0, and, therefore, do not show the rate constant-pH profile that seems general for the redox reactions of this protein. In this work, we studied the rate of reduction of this metalloprotein from pH 1.0 to 6.2. By changing the anionic composition of the reduction medium from chloride to perchlorate, we could measure the reaction rates of the different electronic isomers of the protein.
Experimental Section Materials. Sigma Horse heart cytochrome c Type I l l was used without further purification. (Ru(NH3)6CI3(Johnson Matthey) was purified as follows: I g o f the commercial sample was dissolved in M hydrochloricacid (40 mL). Any solid impurity was filtered off, and the filtrate was cooled to 0 OC in an ice bath. An equal volume of chilled concentrated hydrochloric acid was added to the cold filtrate. whereby the Ru(NH3)6C13 was precipitated. The resulting precipitate was filtered and recrystallized twice from M HCI. The final purified product was air-dried. Ru(NH3)6‘+ was prepared from the purified Ru(NH3)bCI) by zinc amalgam reduction in an anaerobic atmosphere maintained by bubbling argon gas previously scrubbed in chromous towers, to remove the last traces of oxygen. The concentrations of R u ( N H 3 ) h Z + ions were determined by adding excess acidic solutions of iron(1ll) to a known volume of the Ru(NH,)b*+ solution, The iron(ll) formed was complexed with 1 ,IO-phenanthroline, and the concentration of the resulting Fe(phen)3’+ was determined spectrophotometricallyfrom its absorbance at 5 I O nm where its extinction coefficient is I , I X IO4 M-’ cm-’. Trifluorosulfonic acid ( B D H ) was doubly distilled under reduced pressure before use. CF3S03Li was prepared by neutralization of Li2C03 with CF3S03H; dissolved carbon dioxide was removed by bubbling argon into the CFjS03Li solution a t 70 OC. The concentration of the stock solution of CF3S03Li was determined by passing a n aliquot of the solution through a cation exchange column (Amberlite IR 120(H)),and the total acid content of the eluant was determined by titration with standard base. Sodium perchlorate ( B D H A R Grade) was purified by recrystallization from water. Sodium chloride, sodium acetate, hydrochloric acid, and perchloric acid were all BDH ( A R Grade) and wcre used without further purification.Triply distilled water was used in preparing all solutions.
0 1980 American Chemical Society
Adegite. Okpanachi
/
2833
R u ( N H ~ ) Reduction ~~+ of Horse Heart Cyt c
Table 1. Second-Order Rate Constants for the Reaction of Cytochrome c with R U ( N H I ) ~ at ~ +Different pHsa
DH I .o I .2 I .5 I .7 I .9
2.0 2.2 2.7 3.0 3.2 3.35 3.65 3.70 3.8 4.0 4.3 5.0 5.2 5.3 5.4 6.15
1 0 - 4 ~ .M-1
0.30 0.34 1.17 I .75 1.73 3.90 4.65 5.75 6.75 8.29 7.27 7.19 6.71 6.03 5.10 4.50 4.08 3.79 3.80
3.75 3.82
buffer
S-I
NaCl NaCl NaCl NaCl NaCl NaCl NaCl
-
+ HCI + HCI + HCI + HCI + HCI + HCI + HCI
'U
.Lo.. 8 2
sodium formate sodium formate sodium formate
sodium formate/sodium sodium formate/sodium sodium formate/sodium sodium formate/sodium sodium acetate sodium acetate sodium acetate
acetate
acetate acetate acetate
sodium acetate sodium acetate
sodium acetate sodium acetate
[ R u ( N H ~ ) ~ ' =+ ](0.5-4.0) X IO-3 M. [Cytochrome c ] = 3-6 p \ l , All rate constants were obtained at X 550 nm; I = ionic strength = 0.10 M NnCI: t = 25 OC; buffer concentration = I O m M . kinetics. Rates of reaction were measured at 25 "C by monitoring the absorbance changes due to ferrocytochrome c a t 550 nm using a Durrum I I O stopped-flow spectrophotometer. The rate data were obtained by analyz,ing Polaroid photographs of absorbance-time curves recorded on the oscilloscope. For each run, at least three of such traces h e r e analyzed. The temperature of the reaction medium was maintained constant at 25.0 rt 0.1 OC by passing water from a therniostated bath through the cell compartment of the spectrophotometer. In order to avoid kinetic complications arising from sudden changes in ionic strength or pH of the protein solution as reported by previous workers, the ionic strength and pH of both reactants were adjusted to the same values (0.1 M ) before rate measurements were made. All kinetic measurements were made under pseudo-first-order conditions with the concentration of Ru(NH3)6*+ at least 50-500 times that of t h e protein. The concentration of cytochrome c was determined by dissolving known weights of the protein in a known volu m e of the solution.
Results Spectra, The spectral data obtained are in excellent agreement with those previously reported.*." Thus, at pH 5.2 ( I = 0.1 M NaCI) the protein exists in its native low-spin form with a peak a t 530 nm; a t pH 24.0 both the mixed-spin and the low-spin isomers are present. At pH 1 .O ( I = 0.1 M NaCI), the high-spin isomer is dominant with a peak a t 620 nm, and the amount of mixed-spin species has decreased significantly. I n perchlorate acid medium, the spectra show that a t pH 1.7 both the low-spin and mixed-spin forms are present, but as the pH decreases to 1 .O, the amount of the high-spin species increases and the low-spin species decreases. Reduction of Ferricytochrome in 0.1 M Chloride. The kinetics of the reduction of ferricytochrome c were studied at an ionic strength of 0.10 M NaCl and from pH I .O to 6.2. Above pH 2, the first-order plots obtained from the absorbance-time data were linear for more than 90% reaction. The pseudofirst-order rate constants obtained from such plots varied linearly with ruthenium( 11) concentration (0.5 X 1 0-3 to 4.0 X IO-) M). The calculated second-order rate constants varied with pH. The second-order rate constants are presented in Table I . The rates were also found to be independent of buffer concentrations in the range 5.0-10.0 m M for acetate (pH 3.5-6. IS) and formate (pH 2.7-3.8) buffer. Between pH 3.5 and 3.8, the rate constants were insensitive to the buffer medium.
Figure 1. Variation of pseudo-first-order rate constant with R U ( N H J ) ~ ~ + concentration in acid medium. [H+] = 0.1 M = 0.10 M NaCI: t = 25
"C.
(I 0 M
-
Qo
29
I
60
41)
PH
Figure 2. Variation of k with pH for the reaction of R u ( N H ~ ) ~w i~t h+ cytochrome c. Experimental points are in circles. The curve was based on rate constants calculated with eq I : (0) points obtained by Bennett and Ewall.16 I = 0.10 M NaCI, t = 25 "C; (0)HCI NaCl medium: ( 0 ) sodium formate buffer: ( 0 )acetate buffer.
+
At p H 3-5 X I O 5 M-I s-l. It therefore follows from the above that cyt c I , which is a singly protonated and a more readily protonated species than the native protein, is the most reactive form of cytochrome c i n solution, and it is responsible for the observed maximum at about pH 3. The rate data obtained for the range 0.02-0.1 M acid in perchlorate medium can be interpreted in terms of Scheme 11, where cyt c z and cyt c 3 are different conformations of the protonated cytochrome protein. I f we make n steady-state approximation for cyt c3, we obtain for the rate expression, provided the first equilibrium is fast compared to the redox reaction: 1
d dt
- - [cyt c"']
X [cyt c"'][Ru(ll)]
+
(4)
+
where [cyt c1I1]= [cyt c ' ] [cyt c'] [cyt c i ] and Kl = [cyt r ' ] [ H + ] / [ c y t c2]. I f k2 is small and k32 >> k3[Ru(Il)], then this simplifies the expression for the second-order rate constant to: K i k i / [ H + ] + kjK23 (5) Kl/[H+] 1 K23 where K23 = k23/k32. If Kl/[H+]
