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  • Title: Elucidating the mechanism of nucleotide-dependent changes in the redox potential of the [4Fe-4S] cluster in nitrogenase iron protein: the role of phenylalanine 135.
    Author: Ryle MJ, Lanzilotta WN, Seefeldt LC.
    Journal: Biochemistry; 1996 Jul 23; 35(29):9424-34. PubMed ID: 8755721.
    Abstract:
    Nucleotide binding to the nitrogenase iron (Fe) protein results in a lowering of the redox potential of its [4Fe-4S] cluster by over 100 mV, and this is thought to be essential for electron transfer to the molybdenum-iron (MoFe) protein for substrate reduction. This work presents evidence for an important role of the strictly conserved phenylalanine at position 135, located near the [4Fe-4S] cluster of nitrogenase Fe protein, in defining both the redox potential and the nucleotide-induced changes in the redox potential of the [4Fe-4S] cluster. Phe 135 was changed by means of site-directed mutagenesis to the amino acids Tyr (F135Y), Ile (F135I), Trp (F135W), and His (F135H), and the altered proteins were purified to homogeneity. Minor changes in the UV/visible and EPR spectra arising from the [4Fe-4S] cluster were detected in the altered proteins, while dramatic changes were observed in the visible region circular dichroism (CD) spectrum, suggesting that Phe 135 contributes significantly to the chiroptical properties of the [4Fe-4S] cluster. Likewise, significant changes in the redox potentials of the Phe altered Fe proteins were observed, with shifts of +50 to +120 mV compared to the redox potential of the wild-type Fe protein (-300 mV). The shifts in redox potential for the altered Fe proteins appeared to correlate with changes in isotropically shifted proton NMR resonances assigned to cluster ligands. All of the Phe 135 altered Fe proteins were found to bind either MgADP or MgATP, while the reduced and oxidized states of the F135W and F135H altered Fe proteins had significantly higher affinities for binding MgATP when compared to the wild-type Fe protein. While MgATP binding to the wild-type and Phe 135 altered Fe proteins resulted in approximately -100 mV shifts in the redox potentials for all proteins, MgADP binding resulted in only -30 to -50 mV shifts for the altered proteins compared to a -160 mV shift for the wild-type Fe protein. The current results suggest that Phe 135 is important in defining the redox potential of the [4Fe-4S] cluster in the Fe protein and influences the MgADP (but not MgATP) induced modulation of the redox potential.
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