Purification, inter-protein binding and electron transfer reactions in the alkane hydroxylase system of Pseudomonas oleovorans
The production of the recombinant forms of rubredoxin and rubredoxin reductase from Escherichia coli TG1 strain carrying the plasmids pKR10 (1-iron double domain rubredoxin; 1FeDDR), pKR12 (C-terminal domain rubredoxin; CTDR), pKR14 (N-terminal domain rubredoxin; NTDR) and pKRR5V (rubredoxin reductase) in large quantities is described. These proteins were investigated in terms of their binding interactions and electron transfer properties using UV/visible spectroscopy, fluorescence spectroscopy, stopped-flow techniques and temperature-jump techniques. A method to produce stable NTDR has been developed. The protein is stable for several hours at room temperature, and is thus suitable for spectroscopic and kinetics studies. Fluorescence studies were performed exploiting the quenching of FAD fluorescence by rubredoxin on binding to rubredoxin reductase. Electrostatic forces do not drive the interaction between rubredoxin and rubredoxin reductase and the interaction surface is thus likely to be hydrophobic in nature. The dissociation constants obtained for 1FeDDR, 2FeDDR and CTDR were similar. The stopped-flow fluorescence studies showed that the binding of rubredoxin (1FeDDR, CTDR) to rubredoxin reductase occurs in less than 1ms. Further, this study showed a slower phase in the kinetics of interaction representing adjustment of the complex to a more stable form. Complex formation is therefore a multi-step process. Consequently, electron transfer from the reduced rubredoxin reductase to the rubredoxin occurs in a metastable-bound state. The temperature-jump technique, which exploits the perturbation of equilibrium, revealed the observed dissociation rates of CTDR and 1FeDDR from rubredoxin reductase to be around 3000 -1. The kinetics of electron transfer from NADH-reduced rubredoxin reductase to CTDR was analysed using a sequential stopped-flow technique and showed a hyperbolic dependence of reaction rates on rubredoxin concentration with a limiting rate of 61.2 +/- 1.4 s-1 for rubredoxin reductase and dissociation constant for the complex of 12.7 +/- 1.1 mM. CTDR, 1FeDDR and 2FeDDR were found to oxidise the NADH-reduced rubredoxin reductase with approximately equal facility.