Evidence that the Streptomyces developmental protein WhiD, a member of the WhiB family, binds a 4Fe-4S cluster

Piotr Jakimowicz, Myles R. Cheesman, William R. Bishai, Keith F. Chater, Andrew J. Thomson, Mark J. Buttner

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WhiD is required for the late stages of sporulation in the Gram-positive bacterium Streptomyces coelicolor. WhiD is a member of the WhiB-like family of putative transcription factors that are present throughout the actinomycetes but absent from other organisms. This family of proteins has four near-invariant cysteines, suggesting that these residues might act as ligands for a metal cofactor. Overexpressed WhiD, purified from Escherichia coli, contained substoichiometric amounts of iron and had an absorption spectrum characteristic of a [2Fe-2S] cluster. After Fe-S cluster reconstitution under anaerobic conditions, WhiD contained similar to4 iron atoms/monomer and similar amounts of sulfide ion and gave an absorption spectrum characteristic of a [4Fe-4S] cluster. Reconstituted WhiD gave no electron paramagnetic resonance signal as prepared but, after reduction with dithionite, gave an electron paramagnetic resonance signal (g similar to2.06, 1.94) consistent with a one-electron reduction of a [4Fe-4S](2+) cluster to a [4Fe-4S](1+) state with electron spin of S = 1/2. The anaerobically reconstituted [4Fe-4S] cluster was oxygen sensitive. Upon exposure to air, absorption at 410 and 505 nm first increased and then showed a steady decrease with time until the protein was colorless in the near UV/visible region. These changes are consistent with an oxygen-induced change from a [4Fe-4S] to a [2Fe-2S] cluster, followed by complete loss of cluster from the protein. Each of the four conserved cysteine residues, Cys-23, -53, -56, and -62, was essential for WhiD function in vivo.
Original languageEnglish
Pages (from-to)8309-8315
Number of pages7
JournalJournal of Biological Chemistry
Issue number9
Publication statusPublished - 2005

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