Redox reactions of the iron-sulfur cluster in a ribosomal RNA methyltransferase, RumA: optical and EPR studies.
An unprecedented [4Fe-4S] iron-sulfur cluster was found in RumA, the enzyme that methylates U1939 in Escherichia coli 23 S ribosomal RNA (Agarwalla, S., Kealey, J. T., Santi, D. V., and Stroud, R. M. (2002) J. Biol. Chem. 277, 8835-8840; Lee, T. T., Agarwalla, S., and Stroud, R. M. (2004) Structure 12, 397-407). Methyltransferase reactions do not involve a redox step. To understand the structural and functional roles of the cluster in RumA, we have characterized redox reactions of the iron-sulfur cluster. As isolated aerobically, RumA exhibits a visible absorbance maximum at 390 nm and is EPR silent. It cannot be reduced by anaerobic additions of dithionite. Photoreduction by deazariboflavin/EDTA gives EPR spectra, the quantity (56% of S = 1/2 species) and details (g(av) approximately 1.96-1.93) of which indicate a [4Fe-4S](1+) cluster in the reduced RumA. Oxidation of RumA by ferricyanide leads to loss of the 390-nm band and appearance of lower intensity bands at 444 and 520 nm. EPR spectra of ferricyanide-oxidized RumA show a fraction (<8%) of the FeS cluster trapped in the [3Fe-4S](1+) form (g(av) approximately 2.011) together with unusual radical-like spectrum (g' values 2.015, 2.00, and 1.95). RumA also reacts with nitric oxide to give EPR spectra characteristic of the protein-bound iron dinitrosyl species. Oxidation of the cluster leads to its decomposition and that could be a mechanism for regulating the activity of RumA under conditions of oxidative stress in the cell. Sequence data base searches revealed that RumA homologs are widespread in various kingdoms of life and contain a conserved and unique iron-sulfur cluster binding motif, CX(5)CGGC.
Agarwalla
Sanjay
S
Department of Biochemistry and Biophysics, University of California-San Francisco, San Francisco, CA 94107, USA. sanjay@cgl.ucsf.edu
Stroud
Robert M
RM
Gaffney
Betty J
BJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005292:Ferricyanides / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008027:Light; D008280:Magnetics; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D005292:Ferricyanides; D007506:Iron-Sulfur Proteins; C093591:RumA protein, Bacteria; C007931:hexacyanoferrate III; D012313:RNA; D004247:DNA; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase; D010100:Oxygen