Preparation, characterization and intramolecular electron-transfer studies of ruthenium-modified cytochromes c
<p>Redox-active ruthenium complexes have been covalently attached to the surface of a series of natural, semisynthetic and recombinant cytochromes c. The protein derivatives were characterized by a variety of spectroscopic techniques. Distant Fe^(2+) - Ru^(3+) electronic couplings were extr...
| Summary: | <p>Redox-active ruthenium complexes have been covalently attached to the surface
of a series of natural, semisynthetic and recombinant cytochromes c. The protein
derivatives were characterized by a variety of spectroscopic techniques. Distant Fe^(2+) -
Ru^(3+) electronic couplings were extracted from intramolecular electron-transfer rates in
Ru(bpy)_2(im)HisX (where X= 33, 39, 62, and 72) derivatives of cyt c. The couplings
increase according to 62 (0.0060) < 72 (0.057) < 33 (0.097) < 39 (0.11 cm^(-1)); however,
this order is incongruent with histidine to heme edge-edge distances [62 (14.8) > 39
(12.3) > 33 (11.1) > =72 (8.4 Å)]. These results suggest the chemical nature of the
intervening medium needs to be considered for a more precise evaluation of couplings.
The rates (and couplings) correlate with the lengths of a-tunneling pathways comprised
of covalent bonds, hydrogen bonds and through-space jumps from the histidines to the
heme group. Space jumps greatly decrease couplings: one from Pro71 to Met80 extends
the σ-tunneling length of the His72 pathway by roughly 10 covalent bond units.
Experimental couplings also correlate well with those calculated using extended Hiickel
theory to evaluate the contribution of the intervening protein medium. </p>
<p>Two horse heart cyt c variants incorporating the unnatural amino acids (S)-2-
amino-3-(2,2'-bipyrid-6-yl)-propanoic acid (6Bpa) and (S)-2-amino-3-(2,2'-bipyrid-4-yl)propanoic
acid ( 4Bpa) at position 72 have been prepared using semisynthetic protocols.
Negligible perturbation of the protein structure results from this introduction of unnatural
amino acids. Redox-active Ru(2,2'-bipyridine)_2^(2+) binds to 4Bpa72 cyt c but not to the
6Bpa protein. Enhanced ET rates were observed in the Ru(bpy)_2^(2+)-modified 4Bpa72 cyt
c relative to the analogous His72 derivative. The rapid (< 60 nanosecond)
photogeneration of ferrous Ru-modified 4Bpa72 cyt c in the conformationally altered
alkaline state demonstrates that laser-induced ET can be employed to study
submicrosecond protein-folding events. </p>
|
|---|