Spectroscopic and Functional Characterization of Iron–Sulfur Cluster-Bound Forms of Azotobacter vinelandiiNifIscA
2012
Mapolelo, Daphne T. | Zhang, Bo | Naik, Sunil G. | Huynh, Boi Hanh | Johnson, Michael K.
The mechanism of [4Fe-4S] cluster assembly on A-type Fe–S cluster assembly proteins, in general, and the specific role of ᴺⁱᶠIscA 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 vinelandiiᴺⁱᶠIscA, and the ability of ᴺⁱᶠIscA to accept clusters from NifU and to donate clusters to the apo form of the nitrogenase Fe-protein. The results show that ᴺⁱᶠIscA 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 O₂-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⁺-ᴺⁱᶠIscA was competent for rapid activation of apo-nitrogenase Fe protein under anaerobic conditions. Apo-ᴺⁱᶠIscA 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.
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