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J. Biol. Chem., Vol. 279, Issue 33, 34123-34129, August 13, 2004

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Redox Reactions of the Iron-Sulfur Cluster in a Ribosomal RNA Methyltransferase, RumA

OPTICAL AND EPR STUDIES^*

Sanjay Agarwalla, Robert M. Stroud, and Betty J. Gaffney¶||

From the Department of Biochemistry and Biophysics, University of California, San Francisco, California 94107 and the ^¶Biological Sciences Department, Florida State University, Tallahassee, Florida 32306

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 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 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, CX₅CGGC.

Received for publication, May 21, 2004

^* This work was supported by National Institutes of Health Grants GM65268 (to B. J. G.) and GM51232 (to R. M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence may be addressed: Dept. of Biochemistry and Biophysics, University of California, San Francisco, CA 94107. Tel.: 415-476-3937; Fax: 415-476-1902; E-mail: sanjay{at}cgl.ucsf.edu. || To whom correspondence may be addressed: Biological Sciences Department, Florida State University, Tallahassee, FL 32306. Tel.: 850-644-8547; E-mail: gaffney{at}bio.fsu.edu.

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