Expression and characterization of recombinant Rhodocyclus tenuis high potential iron-sulfur protein

The high potential iron-sulfur protein (HiPIP) from Rhodocyclus tenuis stra in 2761 has been overproduced in Escherichia coli from its structural gene, purified to apparent homogeneity, and then characterized by an array of me thods. UV-visible spectra of the reduced and oxidized recombinant protein w ere similar to those of the native protein. EPR of the oxidized protein sho ws g values of 2.11, 2.03, and 2.03. ESI-MS gave a mass difference of 350 D a between the holoprotein and acid-treated protein, consistent with incorpo ration of a [Fe4S4] cluster in the holoprotein, The observed mass of the ap oprotein was 6296.6 Da compared to the expected average molecular mass of 6 297.2 Da of the apoprotein, The reduction potential was determined using cy clic and square-wave voltammetry to be 321 and 314 mV versus NHE, respectiv ely. All the observed properties of the recombinant protein parallel those of the native protein or those of native HiPIPs in general, indicating corr ect folding and incorporation of the iron-sulfur cluster. (C) 2000 Academic Press.