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Biochemistry. 2018 Sep 25;57(38):5533-5543. doi: 10.1021/acs.biochem.8b00823. Epub 2018 Sep 13.

Bfd, a New Class of [2Fe-2S] Protein That Functions in Bacterial Iron Homeostasis, Requires a Structural Anion Binding Site.

Author information

1
Department of Chemistry , University of Kansas , Multidisciplinary Research Building, 2030 Becker Drive , Lawrence , Kansas 66047 , United States.
2
Department of Chemistry , Louisiana State University , 229A Choppin Hall , Baton Rouge , Louisiana 70803 , United States.
3
Protein Structure Laboratory, Del Shankel Structural Biology Center , University of Kansas , 2034 Becker Drive , Lawrence , Kansas 66047 , United States.
4
IMCA-CAT , Hauptman Woodward Medical Research Institute , 9700 South Cass Avenue, Building 435A , Argonne , Illinois 60439 , United States.

Abstract

Mobilization of iron from bacterioferritin (BfrB) requires specific interactions with a [2Fe-2S] ferredoxin (Bfd). Blocking the BfrB:Bfd interaction results in irreversible iron accumulation in BfrB and iron deficiency in the cytosol [Eshelman, K., et al. (2017) Metallomics 9, 646-659]. The only known Bfd structure, which was obtained in complex with BfrB (Protein Data Bank entry 4E6K ), indicated a new fold and suggested that the stability of Bfd is aided by an anion binding site consisting of R26, R29, and K46. We investigated the Bfd fold using site-directed mutagenesis, X-ray crystallography, and biochemistry in solution. The X-ray structure, which is nearly identical to that of Bfd in the BfrB:Bfd complex, shows that the [2Fe-2S] cluster preorganizes residues at the BfrB:Bfd interface into a structure complementary to the Bfd binding site on BfrB. Studies in solution showed rapid loss of the [2Fe-2S] cluster at a low ionic strength but higher stability with an increasing ionic strength, thus supporting a structural anion binding site. Structures of the R26E and R26E/K46Y mutants are nearly identical to that of Bfd, except for a new network of hydrogen bonds stabilizing the region encompassing the former anion binding site. The stability of the R26E and R26E/K46Y mutants, which is weakly and completely independent of solution ionic strength, respectively, corroborates that Bfd requires an anion binding site. The mutations, which caused only small changes to the strength of the BfrB:Bfd interaction and mobilization of iron from BfrB, indicate that the anion binding site in Bfd serves primarily a structural role.

PMID:
30183257
PMCID:
PMC6540754
[Available on 2019-09-25]
DOI:
10.1021/acs.biochem.8b00823
[Indexed for MEDLINE]

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