Electron-transfer studies with the CuA domain of Thermus thermophilus cytochrome ba_3

Abstract

Flash photolysis has been used to initiate electron transfer from excited tris(2,2′-bipyridyl)ruthenium(II) to the Cu_A site of a soluble domain from subunit II of Thermus thermophilus cytochrome ba_3. Luminescence quenching of the excited state of the ruthenium(II) complex was observed at low protein concentrations (20–200 μM Cu_A domain), with second-order rate constants of 2.9 × 10^9 and 1.3 × 10^9 M^(−1) s^(−1) at low and high ionic strength, respectively. Transient absorption measurements demonstrate that 10–20% of the quenching arises from electron transfer (ET). At high protein concentrations (>250 μM Cu_A) and low ionic strength (5 mM Tris, pH 8.1), the quenching rate saturates due to ground-state complex formation; a first-order rate constant of 1.5 × 10^5 s^(−1) was estimated for ET in the complex. Given the high driving forces involved (ΔG° = 1.1 eV), it is possible that these ET reactions occur in the inverted driving-force regime. Spectroscopic measurements indicate that the T. thermophilus Cu_A domain and horse heart cytochrome c form a complex at low ionic strength, with an apparent dissociation constant K_d ∼ 5 μM.

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