NikA binds heme: a new role for an Escherichia coli periplasmic nickel-binding protein.
NikA is a periplasmic binding protein involved in nickel uptake in Escherichia coli. NikA was identified as a heme-binding protein in the periplasm of anaerobically grown cells overexpressing CydDC, an ABC transporter that exports reductant to the periplasm. CydDC-overexpressing cells accumulate a heme biosynthesis-derived pigment, P-574. For further biochemical and spectroscopic analysis, unliganded NikA was overexpressed and purified. NikA was found to comigrate with both hemin and protoporphyrin IX during gel filtration. Furthermore, tryptophan fluorescence quenching titrations demonstrated that both hemin and protoporphyrin IX bind to NikA with similar affinity. The binding affinity of NikA for these pigments (Kd approximately 0.5 microM) was unaltered in the presence and absence of saturating concentrations of nickel, suggesting that these tetrapyrroles bind to NikA in a manner independent of nickel. To test the hypothesis that NikA is required for periplasmic heme protein assembly, the effects of a nikA mutation (nikA::Tn5, Km(R) insertion) on accumulation of P-574 by CydDC-overexpressing cells was assessed. This mutation significantly lowered P-574 levels, implying that NikA may be involved in P-574 production. Thus, in the reducing environment of the periplasm, NikA may serve as a heme chaperone as well as a periplasmic nickel-binding protein. The docking of heme onto NikA was modeled using the published crystal structure; many of the predicted complexes exhibit a heme-binding cleft remote from the nickel-binding site, which is consistent with the independent binding of nickel and heme. This work has implications for the incorporation of heme into b- and c-type cytochromes.
Shepherd
Mark
M
Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
Heath
Mathew D
MD
Poole
Robert K
RK
D018528:ATP-Binding Cassette Transporters / Q000302:isolation & purification / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D008958:Models, Molecular; D009532:Nickel / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D029968:Escherichia coli Proteins; C089849:nikA protein, E coli; D006418:Heme; D009532:Nickel; D014364:Tryptophan