INTRAMOLECULAR ELECTRON-TRANSFER IN SINGLE-SITE-MUTATED AZURINS

Single-site mutants of the blue, single-copper protein, azurin, from P seudomonas aeruginosa were reduced by CO2- radicals in pulse radiolysi s experiments. The single disulfide group was reduced directly by CO2- with rates similar to those of the native protein [Farver, O., & Pech t, I. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6968-6972]. The RSSR- r adical produced in the above reaction was reoxidized in a slower intra molecular electron-transfer process (30-70 s-1 at 298 K) concomitant w ith a further reduction of the Cu(II) ion. The temperature dependence of the latter rates was determined and used to derive information on t he possible effects of the mutations. The substitution of residue Phe1 14, situated on the opposite side of Cu relative to the disulfide, by Ala resulted in a rate increase by a factor of almost 2. By assuming t hat this effect is only due to an increase in driving force, lambda = 135 kJ mol-1 for the reorganization energy was derived. When Trp48, si tuated midway between the donor and the acceptor, was replaced by Leu or Met, only a small change in the rate of intramolecular electron tra nsfer was observed, indicating that the aromatic residue in this posit ion is apparently only marginally involved in electron transfer in wil d-type azurin. Pathway calculations also suggest that a longer, throug h-backbone path is more efficient than the shorter one involving Trp48 . The former pathway yields an exponential decay factor, beta, of 6.6 nm-1. Another mutation, raising the electron-transfer driving force, w as produced by changing the Cu ligand Met121 to Leu, which increases t he reduction potential by 100 mV. However, the increase in rate was le ss than expected from the larger driving force and is probably compens ated by a small increase in lambda. Marcus theory analysis shows that the observed rates are in accordance with a through-bond electron-tran sfer mechanism.