Characterization of a Ferredoxin from Desulfovibrio Vulgaris (Hildenborough) that Interacts with RNA

The purification and characterization of a ferredoxin from Desulfovibrio vulgaris (Hildenborough) is described. The protein can be isolated in two forms; the major form is strongly complexed to RNA, while a minor form is free from nucleic acid. Bound RNA cannot be removed by digestion with nucleases, or by heating to 70 °C, and it can only be partially removed by rechromatography. The ultraviolet/visible spectrum shows typical absorption maxima at 280 nm and 400 nm for the RNA‐free ferredoxin. The RNA‐bound protein exhibits an additional strong peak at 260 nm. The RNA can be extracted from the protein with phenol. The ferredoxin is a dimer of subunits, each of 7.5 kDa; its pI is 3.9. The protein contains a [4Fe–4S]( 2+;1+ ) cluster with an EPR spectrum ( g = 1.90, 1.93 and 2.05) in the reduced state. A reduction potential of – 360 mV was determined for the RNA‐free ferredoxin with reversible voltammetry at glassy carbon. From the temperature dependence of the reduction potential, the unusually high standard reaction entropy was calculated as ? S ˚=– 230 J · K ‐1 · mol ‐1 . No electrochemical response was obtained with the RNA‐bound ferredoxin. Binding of RNA appears to require the presence of an intact cluster, since the absence of absorption at 400 nm runs in parallel with the absence of absorption at 260 nm. The possibility is discussed that the binding to the RNA has a regulatory function and is controlled by the state of the cluster.