Characterization and reconstitution of Pseudomonas ovalis ferredoxin

Abstract

A ferredoxin isolated from Pseudomonas ovalis was characterized by spectroscopic experiments. P. ovalis ferredoxin contained 6–8 moles of non-heme iron and acid-labile sulfur atoms based on the molecular weight of 11 900 calculated from the amino-acid sequence (Hase, T., Wakabayashi, S., Matsubara, H., Ohmori D. and Suzuki K. (1978) FEBS Lett. 91, 315–319). The ferredoxin had the ability to replace spinach ferredoxin in the reduction of cytochrome c with spinach ferredoxin-NADP + reductase and NADPH. The absorption maximum of ferredoxin was at 283 mm and shoulders were at 320 and at 400 nm. The ferrodoxin exhibited a circular dichoism (CD) spectrum with positive peaks at 227, 390, 466 and 570 nm. A relatively isotropic EPR spectrum was observed at g = 2.01 in isolated form: the EPR spectrum was characteristic of a three-iron Fe-S center. A reconstituted ferredoxin was made by incubating apoprotein with Fe 2+ and S 2− in the presence of dithiothreitol. The reconstituted ferredoxin had similar properties to those of the native ferredoxin with respect to the ability of cytochrome c reduction, absorption spectrum, and CD spectrum. The reconstituted ferredoxin exhibited an EPR spectrum which was characteristic of a three-iron FeS center; a three-iron center was rebuilt by the reconstitution.