title: Spectroscopic and functional characterization of iron-sulfur cluster-bound forms of Azotobacter vinelandii (Nif)IscA.
abstract: The mechanism of [4Fe-4S] cluster assembly on A-type Fe-S cluster assembly proteins, in general, and the specific role of (Nif)IscA in the maturation of nitrogen fixation proteins are currently unknown. To address these questions, in vitro spectroscopic studies (UV-visible absorption/CD, resonance Raman and Mössbauer) have been used to investigate the mechanism of [4Fe-4S] cluster assembly on Azotobacter vinelandii(Nif)IscA, and the ability of (Nif)IscA to accept clusters from NifU and to donate clusters to the apo form of the nitrogenase Fe-protein. The results show that (Nif)IscA can rapidly and reversibly cycle between forms containing one [2Fe-2S](2+) and one [4Fe-4S](2+) cluster per homodimer via DTT-induced two-electron reductive coupling of two [2Fe-2S](2+) clusters and O(2)-induced [4Fe-4S](2+) oxidative cleavage. This unique type of cluster interconversion in response to cellular redox status and oxygen levels is likely to be important for the specific role of A-type proteins in the maturation of [4Fe-4S] cluster-containing proteins under aerobic growth or oxidative stress conditions. Only the [4Fe-4S](2+)-(Nif)IscA was competent for rapid activation of apo-nitrogenase Fe protein under anaerobic conditions. Apo-(Nif)IscA was shown to accept clusters from [4Fe-4S] cluster-bound NifU via rapid intact cluster transfer, indicating a potential role as a cluster carrier for delivery of clusters assembled on NifU. Overall the results support the proposal that A-type proteins can function as carrier proteins for clusters assembled on U-type proteins and suggest that they are likely to supply [2Fe-2S] clusters rather than [4Fe-4S] for the maturation of [4Fe-4S] cluster-containing proteins under aerobic or oxidative stress growth conditions.
authors.affiliation: Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA.
authors.forename: Daphne T
authors.initials: DT
authors.lastname: Mapolelo
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
authors.forename: Sunil G
authors.initials: SG
authors.lastname: Naik
authors.forename: Boi Hanh
authors.initials: BH
authors.lastname: Huynh
authors.forename: Michael K
authors.initials: MK
authors.lastname: Johnson
mesh_terms: D016948:Azotobacter vinelandii / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D004229:Dithiothreitol / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D015204:Spectroscopy, Mossbauer; D013059:Spectrum Analysis, Raman
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D004229:Dithiothreitol