MECHANISMS OF ENZYMATIC REACTIONS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 50

media inside protein globules, the rough estimation gave for the dielectric

constant
Concerning proteins, the value is strongly dependent on local polarity, which differs
in different portions of such a mosaic structure as a protein globule. Positions of the donor
and acceptor centers relative to the protein-water interface, chemical nature and mobility of
adjacent groups can drastically affect values. Thus, the precise calculation of real in

biological objects requires special theoretical approaches.
An effective approach to the systematic studies that are required to explore the
fundamental aspects of ET in proteins, involves measurements of ET in proteins modified
by artificial donor and acceptor centers. By varying redox properties and position of the
centers, it has been possible to elucidate the factors affected the rate of long-range electron
transfer reactions in proteins. A particularly significant contribution in this area has been
made by H. B. Gray and his colleagues using redox metalloproteins that have been surface-
labeled with redox-active ruthenium complexes (Gray and Ellis, 1994; Gray and Winkler,
1996; Tezcan et al., 2001; Ponce et al., 2000; Winlker et al., 1999).
complexes readily react with surface histidine residues to form stable derivatives.
Photochemical methods were used to inject an electron into the

site followed by

monitoring kinetics of ET from to the metalloprotein active site.
The reduction potential can be varied from <0.0 to > 1.5 eV. The values can
be estimated by the analysis of experimental dependences log According to the
Marcus 2.5, the maximum of this dependence is related to the equality Such an
analysis was performed by Gray and Winkler (1996) using data on the driving-force
dependence of long-range electron-transfer rates in Fe-cytochrome c and Zn-substitutes
cytochrome c modified by Ru complexes with different ligands effected on The
estimated values were found to be different for different complexes that highlight the
important role of interaction of complexes with water. The bulky bipyridine ligands shield
the charged metal center from the polar aqueous solution reducing the solvent
reorganization as compared to less bulky ligands These experiments
also demonstrated that centers located in the aqueous phase contribute more significantly
than groups, buried in protein globule.
According to Miyashita and Go, 2000 the main contribution to in electron transfer in
cytochrome c, modified by the complex, originates from the interaction of donor
and acceptor with protein groups and water, whereas the contribution of high-frequency
vibration modes inside of the donor and acceptor centers is very small (about 1%).
Nevertheless, high-vibration modes of protein and water add about 30% to the system
fluctuation energy. This calculation also revealed the strong correlation between protein
and water and, therefore the division of reorganization energies between protein and water
in a simple way is not appropriated. The reorganization energy can be also estimated from
experimental Gibbs energy activation (Eq.2.6) (Fogel et al., 1994) when is known.
A problem of the experimental measurement of local polarity in the vicinity of donor
and acceptor centers incorporated into a protein (bovine serum albumin, BSA) was solved
with the use of the dual fluorescence-nitroxide probe (Bystryak et al., 1986; Rubtsova et
al., 1993; Fogel et al., 1994; Likhtenshtein, 1993, 1996; Likhtenshtein et al., 2001). In such
a hybrid molecule, the photoactive chromophore fragment in the excited singlet state can