Expression and characterization of recombinant Rhodocyclus tenuis high potential iron-sulfur protein.
The high potential iron-sulfur protein (HiPIP) from Rhodocyclus tenuis strain 2761 has been overproduced in Escherichia coli from its structural gene, purified to apparent homogeneity, and then characterized by an array of methods. UV-visible spectra of the reduced and oxidized recombinant protein were similar to those of the native protein. EPR of the oxidized protein shows g values of 2. 11, 2.03, and 2.03. ESI-MS gave a mass difference of 350 Da between the holoprotein and acid-treated protein, consistent with incorporation of a [Fe(4)S(4)] cluster in the holoprotein. The observed mass of the apoprotein was 6296.6 Da compared to the expected average molecular mass of 6297.2 Da of the apoprotein. The reduction potential was determined using cyclic and square-wave voltammetry to be 321 and 314 mV versus NHE, respectively. All the observed properties of the recombinant protein parallel those of the native protein or those of native HiPIPs in general, indicating correct folding and incorporation of the iron-sulfur cluster.
Caspersen
M B
MB
Department of Chemistry, Technical University of Denmark, Building 207, Lyngby, DK-2800, Denmark.
Bennett
K
K
Christensen
H E
HE
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry; D001483:Base Sequence; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis* / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D045322:Photosynthetic Reaction Center Complex Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012245:Rhodospirillales / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D011994:Recombinant Proteins; C007943:high potential iron-sulfur protein