Control mechanisms of intra-molecular electron transfer in oxidoreductases

• Main Author: Turner, Karen L.
• Published: University of Edinburgh 1999
• Subjects: 572.7
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.663113

The intra-molecular electron transfer steps in three multi-centred oxidoreductases were studied by redox potentiometry and protein film voltammetry. Cytochrome <i>cd₁ </i>is a disimmilatory nitrite reductase, the achieves the reduction of nitrite to nitric <i>oxide.</i> Reduction of the enzyme from <i>Thiosphaera pantotropha</i> is associated with a conformational change (Williams <i>et al.,</i> 1997) and hysteresis is observed in the potentiometric titration at room temperature (Kobayashi <i>et al.,</i> 1997). The potentials in the oxidative and reductive directions are separated by 150 mV at 20°C but ?DE decreases at high temperatures. The increased reversibility at higher temperatures is also shown by a rise in the n-value and the appropriate spectral response when the potential is perturbed in both the oxidative and reductive directions. At the highest temperatures, when equilibrium conditions are reached, the interconversion of the completely oxidised and completely reduced states is characterised by a potential of 140 mV and an n-value of 2. At 20 °C the electron transfer is conformationally gated such that a proposed square scheme is essentially a one-way system. As the temperature is raised, the rate of the conformational change increases and the interconversion approaches reversibility within the measurement time. Flavocytochrome P-450 BM3 is a fatty-acid monooxygenase from <i>Bacillus megaterium.</i> The complete redox characterisation of the enzyme has been achieved by potentiometric titrations on the intact holoenzyme and its component domains. The reduction potential of the haem is increased by more than 100 mV on fatty-acid substrate binding at the active site (E_m = -368 ± 6 mV, substrate-free, to E_m = -239 ± 6 mV, arachidonate-bound). The FMN to haem electron-transfer is under thermodynamic control, such that the futile cycling of electrons and hydrogen peroxide formation is prevented. The blue FAD semiquinone species is observed during the course of the reductive titrations of these enzymes (E₁ = -283 mV).