RR'NH + HCOH
Scheme 2 Scheme 2
Figure 4.5. Postulated mechanism of action of Na2EDTA in the flash photolysis experiment (D. Miller, G. McLendon, Inorg. Chern. 20,950 (1981)). EDTA2- (R=CH2COOH, R'=CH2CH2N(CH2COO-h) acts to reduce [Ru(bpy))]3+
(A=acceptor) and prevent its reduction by the protein under study. The radical intermediate is believed to be responsible for the slow reduction of the native cytochrome, i.e., the second equivalent of acceptor may be ferricyto- chrome rather than [Ru(bpy))]3+.
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Table 4.1. [Ru(bpy)3J2+* lifetimes in the presence and absence of tryptophan
sample
[Ru(bpy) 3]2+
[Ru(bpy)3]2+
[Ru(bpy)3]2 + + Trp
591.5 573.7 581.5
a. average of two values, each determined by fits to averages of at least 200 pulses.
potential,9 assuming a value of 0.84V 10 for [Ru(bpy)3]2 +* reduction. 11
Since the reduction potential of a tryptophan residue might be expected to vary depending on its particular protein environment, studies should be conducted to test for the transient formation of the Trp· radical upon [Ru(bpy) 3]2+ excitation and subsequent cytochrome reduction. C. krusei cytochrome c contains a single tryptophan residue (number 59). 12 The absorbance can be monitored at 526.5nm, a
8 Jovanovic, S., Harriman, A., Simic, M. J. Phys. Chern. 90, 1935 (1986).
9 Once oxidized, tryptophan may lose a proton, depending on the solution pH. The pKa has been reported to be 4.3 by Posener, M., Adams, G., Ward man, P., Cundall, R. J.C.S. Faraday Soc. I 72, 2231 (1976).
10 Creutz, C., Sutin, N. Inorg. Chem. 15, 496 (1976).
11 Butler, J., Land, E., Swallow, A., Prlitz, W. J. Phys. Chem. 91, 3114 (1987).
12 Narita, K., Titani, K. J. Biochem. 63, 226 (1968).
C. krusei cytochrome c isosbestic point, near the 485-510nm absorbance maximum of Trp·. 13
In an intramolecular electron transfer experiment such as that described above, where the fully oxidized protein is utilized (i.e., E0(Fe)>E0(Ru)), formation of Trp· would not interfere with the electron transfer rate constant determination provided no more than one bi- molecular redox reaction occurred per cytochrome molecule. However, for other systems, where the fully reduced protein is utilized, Trp·
formation and subsequent Fe2+ -+Trp· electron transfer might be observed in addition to Fe2+-+ Ru3+ electron transfer.
Reduction of amino acid residues was not considered a possibility, as the potentials of the tryptophan, tyrosine, and phenyl- alanine amino acid/radical anion couples have been estimated from preliminary experiments to be more negative than -1 V. 14 A value of -0.84V has been reported for the [Ru(bpy)
3]3+ /[Ru(bpy)
3]2 +* reduction potential. 15
Upon flashing the native C. krusei cytochrome in the presence of [Ru(bpy) 3]2+, a rapid rise in absorbance is observed (figure 4.6).
This confirms that the mechanism of [Ru(bpy)
3]2+* quenching by cyto- chrome is indeed an electron transfer process. Ideally, the excited ruthenium complex would transfer an electron to the heme of native cy- tochrome c, and no further change in absorbance would be observed.
13
14
(a) Redpath, J., Santus, R., Radial. Bioi. 27, 201 (1975);
Photochem. Photobiol. 15, 101 ( 1972).
Ovadia, J., Grossweiner, L. Int.
(b) Santus, R., Grossweiner, J.
L.
Butler, J., Land, E., Pri.itz, W., Swallow, A. Biochim. Biophys. Acta 705, 150 ( 1982).
15 Lin, C., Sutin, N. J. Phys. Chern. 80, 97 (1976).
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Sxl0-3.---.---~----r---~----.---~----.---~~---r----~--~----~
4x10-3
Q) 0 c
0 .D
\....
0
(I)
.D 0 Q) >
·;, 0 Q)
\....
2x 10-3
0~--~--~----~--~----J---~----~----~--~----~--~----~
0 20 40 60 80 100
time (seconds)
Figure 4.6. Flash photolysis data for native C. krusei cytochrome c (A.=549nm). The initial jump in absorbance at the time of the flash (Os) is attributed to reduction of the heme iron by the excited ruthenium(II) tris(bipyridyl) complex. The slower reduction is attributed to EDTA radicals which are formed by reaction with the oxidized ruthenium(III) tris(bipyridyl) complex. The data can be approximated by a monoexponential function (k=0.05/s, an average of three experiments).
However, the data reveal a slow process which can be fitted to a mono- exponential function with a rate constant of -O.OSs- 1 for concentra- tions identical to those at which the modified protein kinetics were studied. The process is believed to be the reduction of the heme iron by the EDT A radicals which
[Ru(bpy)3]3+.
result from the reduction of the
On a similar time scale (IOOs), little absorbance change is observed upon flash photolysis of the modified protein after the initial increase (figure 4.7). Data collected on a much shorter time scale reveal an absorbance increase and leveling after approximately 16ms in addition to the initial jump expected from direct reduction of the iron site (figure 4.8). This process is attributed to the migra- tion of electrons from the ruthenium(II) centers (created upon flashing by oxidation of [Ru(bpy)3]2+*) to the iron(III), and proceeds with a rate constant of -170s- 1. The concentration independence of the ob- served rate over the l-6~M range ([cytochrome]) supports the assumption that this is an intramolecular process. Although EDT A radicals may be present, further absorbance changes are minimal because the protein is almost completely reduced. l6
If the intramolecular electron transfer data is fit beyond 7ms, so as to eliminate that which might be affected by the problem of flash
16 The similar values of the absorbance change for the native and modi- fied samples suggests that a similar number of bimolecular electron transfer events take place. Hence, the EDT A radical concentration is expected to be similar in each case. Direct EDT A radical reduction of the iron site, or rate-determining radical reduction of the ruthe- nium site, followed by fast intramolecular electron transfer, may be responsible for the slow rise observed in the 0-Ss range following flash photolysis of the modified derivative.
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Cl)
0 6x 1
o-
3c 0 ..0 ....
0
Ul ..0 0 Cl)
>
~ 0 4x 1
o-
3Q) ....
0~--~--~---L----~--~----~----~--~~---L----~--~~--~
0 20 40 60 80 100
time (seconds)
Figure 4.7. Flash photolysis of modified C. krusei cytochrome c (A..=549nm).
Reduction is rapid, and little of the observed signal is due to EDTA radical reduction.
6x10-3r-,---~----~----~---r----~--~----~---,----~----r----r----r-•
4x1o-3
Q) 0 c:
0 .D ....
0
1/)
.D 0 Q) >
:g
Q)
.... 2x1 o-3
o~~--~---L~~----L-~~--~~~--~~~~--~--~--~~
2x1o-3 4x1o-3 6x1o-3 8x1o-3
0 0.01 0.012
time (seconds)
Figure 4.8. Data for flash photolysis of the modified C. krusei cytochrome c. The average of monoexponential fits to two runs for the 2-12ms range is 170/s.
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breakthrough, an average rate of 295s- 1 is obtained (figure 4.9). This value is much more uncertain, but as stated previously, 170s- 1 is prob- ably a lower limit to the actual electron transfer rate constant. Rate constants for the intramolecular electron transfer in the ruthenium- modified protein are tabulated in table 4.2.
C) 0 c:
0 .D ....
0
II)
.D 0 Q) >
:;;
0
~ 2x10-3
o~~--~----~~-L----~~~----~~~----~~~----~--~--~~
2x10-3 4x10-3 6x10-J 8x10-3
0 0.01 0.012
time (seconds)
Figure 4.9. Data for flash photolysis of the modified C. krusei cytochrome c. The average of rnonoexponential fits to two runs for the 7-12rns range is 295/s.
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Table 4.2. Monoexponential fits to the kinetic data of modified cytochrome c a
A. t>2ms
parameter
A(1) 0.0096 ± 0.0002 0.0013 ± 0.0001
A(2) 0.0041 ± 0.0001 0.0049 ± 0.0001
194 ± 20 145 ± 16
B. t>7ms
parameter Run 1
Rlin..2
A(1) -0.0027 ± 0.009 -0.0022 ± 0.006
A(2) 0.0075 ± 0.009 0.0078 ± 0.006
k (s-1) 320±210 270 ± 139
a. Data were fit to an equation of the form y