Photochemistry and Radiation Chemistry - American Chemical Society

10 Electron Transfer Kinetics of Bifunctional Redox Protein Maquettes Mitchell W. Mutz , James F. Wishart , and George L. McLendon* 1

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Department of Chemistry, Princeton University, Princeton, NJ 08544 Department of Chemistry, Brookhaven National Laboratory, Upton, NY 11973

Downloaded by UNIV OF PITTSBURGH on March 2, 2016 | http://pubs.acs.org Publication Date: April 17, 1998 | doi: 10.1021/ba-1998-0254.ch010

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We prepared three bifunctional redox protein maquettes based on 12-, 16-, and 20-mer three-helix bundles. In each case, the helix was capped with a Co(III) tris-bipyridyl electron acceptor and also functionalized with a C-terminal viologen (1-ethyl-1'-ethyl-4,4'-hipyridinium) donor. Electron transfer (ET) was initiated by pulse radiolysis andflash photo lysisand followed spectrometrically to determine average, concentra tion-independent,first-order rates for the 16-mer and 20-mer ma quettes. For the 16-mer bundle, the α-helical content was adjusted by the addition of urea or trifluoroethanol to solutions containing the metal -loprotein.This conformational flexibility under different solvent condi tions was exploited to probe the effects of helical secondary structure on ET rates. In addition to describing experimental results from these helical systems, this chapter discusses several additional metalloprotein models from the recent literature.

I n the study of protein electron transfer (ET), radiolytic and photochemical techniques have indeed proven highly complementary. Between them, these techniques provide a range of reaction types and reaction free energies [cf. Zn porphyrin triplets (F° ~ 0.8 V) versus Fe porphyrins (E° ~ 0 V)]. O f particular interest in the current study is the different dynamic range(s) of the techniques. Photochemistry is subject to a natural "time window" set by the excited state lifetime: only reactions faster than the excited state decay can be observed. Conversely, the bimolecular nature of radiolysis sets an upper limit on the observed rates that is often determined by the rate of electron capture. A n early example of these complementary aspects was provided by studies * Corresponding author. ©1998 American Chemical Society

In Photochemistry and Radiation Chemistry; Wishart, James F., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1998.

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PHOTOCHEMISTRY AND RADIATION CHEMISTRY

of the cytochrome c: cytochrome bs complex. Initial studies utilized photochem istry to monitor reactions at high free energy, -aG, and radiolysis to monitor (slower) reactions at lower -AG(l-2). A n interesting sidelight of these earlier studies has emerged. Using an intramolecular ruthenium bipyridyl (bpy) photosystem, Durham, Millett, and co-workers have reinvestigated the cyt b :cyt c system, as reported i n Chapter 7. With the higher time resolution of their photochemical technique, they observe two populations of reactants: a "slow" population reacts with fc b = 3 ± 1 X 10 s , as reported in the earlier study (3). However, they also observe a faster population (k \, = 4 ± 1 Χ 10 s" ). This faster phase occurs on a time scale competitive with electron capture and was therefore missed in the radiolysis study. In retrospect, it appears possible to model the earlier data in terms of a hmiting rise time of the (fast) signal. Assuming steady-state kinetics where the rate of intraeomplex electron transfer is faster than electron capture, the apparent rise time will in fact monitor the rate of electron transfer (and not of electron capture). Such a model indeed appears to reproduce the work of Millett and co-workers (3). A different example of complementarity is explored i n this chapter. W e report studies of a bifunctional redox protein maquette based on the triplehelix bundle design of Ghadiri, Sasaki, and co-workers (4-5). Attempts to moni tor electron transfer from an N-terminal ruthenium tris-bipyridyl excited state were fruidess, since electron transfer could not compete with the excited state decay of the R u ( I P ) tris-bpy. However, as described below, replacement of photoactive ruthenium by a radiolytically accessible Co couple has allowed initial exploration of electron transfer in this synthetic protein couple. In turn, photochemistry initiated at the viologen chromophore helped confirm and ex tend the radiolytic results. 5

G

s

4

-1


0

s

5

1

1

îîmi

Electron Transfer in Synthetic Three-Helix Bundles De novo design of redox proteins represents a significant challenge for biologi cal and biomimetic chemistry (6-8). Several maquettes have been designed toward systems in which electrons can be translocated across proteins (9-11 ). A wealth of data now exists for modified natural proteins like cytochrome c (12-16). Significant data are also available for modified single peptide systems (17-19), but conformational equilibria often complicate the interpretation of simple systems (20). However, detailed analyses of de novo proteins that adopt well-defined conformations remain rare. Two particularly attractive structural maquettes for the design and study of de novo redox proteins were reported by Ghadiri et al. (4) and Lieberman and Sasaki (5). Both systems consist of a three-helix bundle whose stoichiometry and topology are defined by the capping metal tris-bipyridyl complex. These maquettes are designed to maximize interhelical interactions, thereby providing a more stable conformation than is gener-

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All relevant experimental details on this work can be found in reference 21.


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Μυτζ ET AL. Protein Maquettes

147

Ο

ΛNH S

B-N-A-E-Q-L-L-Q-E-A-E-Q-L-C H

\ v

2

O

^NH B-N-A-E-Q-L-L-Q-E-A-E-Q-L-L-Q-E^C H \ v

2

o

Λ

NH B-N-A-E-Q-L-L-Q-E-A-E-Q-L-L-Q-E-A-E-Q-L-L- C H \y X

2


2

/

w

\ ^

-

-

A = L - Alanine E = L - Glutamic Acid Q = L -Glutamine L = L - Leucine C = L - Cysteine

Figure 1. Structures and amino acid sequences of hipyridine-peptide-viologen complexes (bipep-V).

ally available in isolated, single peptides. Additionally, since there are numerous tris-bipyridyl complexes, using this motif to create three-helix bundles allows ready access to the many varied spectroscopic, photophysical, and redox proper ties offered by these metal compounds. A minor elaboration on these maquettes provides a model bifunctional redox system, in which the bipyridine-modified peptides, bipep, are covalendy modi fied with a redox-active viologen at the C-terminus as shown in Figure 1, and a redox-active metal is incorporated into the N-terminus (see Figure 2). Details of the synthesis and purification of these compounds can be found elsewhere (21 ). The 16- and 20-mer bundle complexes, M - P - V , where M is cobalt, iron, or ruthenium complexed with the bipyridine ligands, Ρ is the peptide bridge, and η is the number of residues per single strand, adopt the helical configurations already defined by the parent peptides, as shown in Figure 3 (22). However, the helical content of C o - 1 2 -mer was only 30%, even in the presence of trifluoroethanol (TFE), a known helix-inducing reagent. For this reason, the 12-mer bundles were not used as models for helix-mediated E T in this study. When the metal in these systems is cobalt, the C o tris-bipyridine and violo gen moieties of C o - P „ - V readily undergo redox reactions at similar potentials to those of the isolated systems (Co tris-bipep: E° = +0.15; viologen: E° = -0.64, both versus saturated N a C l calomel electrode (SSCE); p H = 7.0, deter mined by cyclic voltammetry). When the metal is ruthenium, the Ru(II) tris-bi pyridine excited state, Ru(II* ), is expected to have E° —0.8 V (17). n

m

111

m

111



148


Figure 2. Sketch of the putative structure of an M-P -V maquette. The bipyridines are at the top of the page, with the metal center represented as a black sphere. The viologen group is the bicyclic ring at the bottom of the page. n

Ffosh Photolysis of Ruthenium-Modified Helical Maquettes First attempts to measure electron transfer reactions in these maquettes fo cused on the R u - P - V system. A l l ruthenium-modified complexes were pre pared according to standard procedures (4, 23). Time-resolved fluorescence measurements of Ru(II*)-P„ revealed lifetimes of about 490 ns, consistent with those of Ru(2,2'-bipyridine)3 * (24). Based on this lifetime, the only E T rates that would be direcdy measurable by this technique are those faster than about 2 Χ 10 s~ . A possible E T reaction takes place as shown in Scheme I. n

n

+2

6

l



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Μυτζ ET AL. Protein Maquettes

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Figure 3. Circular dichroism spectra of Co-Ρ -V maquettes, where η = 12, 16, and 20 are represented by dotted, solid, and dotted-dashed curves, respectively. As the number of residues increases, the helicity of the bundles is enhanced, as shown by the increased negative ellipticity at 222 nm. All ellipticity measurements are expressed as mean residue ellipticity. The spectra were obtained in 100 mM formate and 50 mM phosphate, pH = 7.0, at 25 V. Peptide quantitation was by amino acid analysis in all cases. n

R II_p _v2+ u

n

h

>

R u

ll*_p _v2+ n

ι ET R i i * _ p _ V 2 + ——> u

n

Ru -P -V . m

n

+

Scheme I. Following the creation of the Ru(II* ) excited state, an electron can, in prin ciple, be transferred from the ruthenium to the viologen moiety. Since there was neither an absorbance change of the viologen moiety (monitored at 600 and 370 nm) nor a change in the fluorescence Ufetime of R u ( l l * ) in the presence of violo gen, it was concluded that &ET was less than the rate of R u ( l l * ) fluorescence decay. As a result of these data, we decided to use pulse radiolysis as a means of accessing slower electron transfer rates which might be evidenced by this system.

Measurements of Cobalt-Modified Systems As mentioned previously, the upper limit on the measurement of a reaction rate in pulse radiolysis is often the addition rate of the electron or radical to the reactant of interest. To measure the addition rate of C 0 ~ to viologen in the bundles, F e - P - V was used. The F e state is inaccessible and the reduction 2

n

n

1



150


Figure 4. Puke radiolysis transient absorption data for Fe-P^-V, illustrating the prompt reaction of C0 ~ with the viologen moieties in the maquette. Note that there is a slow (ca. 40 s' ) decay of the viologen absorption, effectively putting a lower limit on the rate of electron transfer that is observable with this technique. One source of this decay could be small amounts of oxygen in the sample. 2

1

of the bipyridine moiety is slow, allowing one to monitor the viologen absor bance change without interfering chemistry from another redox-active group and determine a second-order rate constant for the production of reduced viologen. The addition rate of C 0 " was found to be 4.8 Χ 10 s" for the F e - P i - V complex at a concentration of 5 μΜ. Since there are, on average, two viologens per complex, the second-order rate constant for viologen reduction is 4.8 X 10 M " s" . Kinetic data are shown in Figure 4. Measurements of electron transfer rates by pulse radiolysis studies of C o - P - V show a diffusion-controlled reduction of the viologen chromophore, followed by exergonic (AG = -0.80 eV) electron transfer to the cobalt, with rate constants of 630 and 360 s~ for the 16-mer and 20-mer complexes, respectively (Figure 5). The reaction proceeds according to Scheme II. The 3

2

2

s

m

1

1

n

l

e"(2 Mev)

^ N 0/HC0 Na 2

• CO

Photochemistry and Radiation Chemistry - American Chemical Society

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