title: Evidence that the Streptomyces developmental protein WhiD, a member of the WhiB family, binds a [4Fe-4S] cluster.
abstract: 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 approximately 4 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 approximately 2.06, 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.
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom. Piotr.Jakimowicz@bbsrc.ac.uk
authors.forename: Piotr
authors.initials: P
authors.lastname: Jakimowicz
authors.forename: Myles R
authors.initials: MR
authors.lastname: Cheesman
authors.forename: William R
authors.initials: WR
authors.lastname: Bishai
authors.forename: Keith F
authors.initials: KF
authors.lastname: Chater
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thomson
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Buttner
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D007477:Ions; D007501:Iron / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008024:Ligands; D008280:Magnetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013053:Spectrophotometry; D013302:Streptomyces / Q000378:metabolism*; D013440:Sulfides / Q000737:chemistry; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
chemical_list: D001426:Bacterial Proteins; D007477:Ions; D007506:Iron-Sulfur Proteins; D008024:Ligands; D013440:Sulfides; D014157:Transcription Factors; C498877:WhiD protein, Streptomyces coelicolor; C081543:whiB protein, Streptomyces; D007501:Iron; D003545:Cysteine; D010100:Oxygen