Section 11
Chapter 10,005

The environment of (2Fe-2S) clusters in ferredoxins: the role of residue 45 probed by site-directed mutagenesis

Vidakovic, M.; Fraczkiewicz, G.; Dave, B.C.

Biochemistry (American Chemical Society) 34: 906-13


DOI: 10.1021/bi00042a023
Accession: 010004685

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The biochemical and biophysical properties of the Ala45Ser mutant of the [2Fe-2S] ferredoxin from vegetative cells of the cyanobacterium Anabaena sp. 7120 are described. This novel protein, which incorporates the residue present in many higher plant ferredoxins into the analogous position of a typical cyanobacterial ferredoxin, was prepared to probe the origin of the characteristic spectrochemical and functional differences between the ferredoxins from these two sources. The variant protein was produced by site-directed mutagenesis and was expressed as the holoprotein in Escherichia coli. Although the UV-vis spectrum of the Ala45Ser mutant was indistinguishable from that of the wild-type (WT) protein, the circular dichroism (CD) spectrum of the mutant was distinct and similar in appearance to that of spinach ferredoxin, which possesses a Ser residue at the analogous position. The values of the principal g factors of the EPR spectrum of the dithionite-reduced mutant protein differed from those of the WT spectrum and resembled those of plant ferredoxins containing serine at position 45. Analysis of the mutant EPR spectrum according to the method of Blumberg indicated greater covalent interactions between the localized ferrous site of the cluster and the protein matrix relative to the WT protein. The resonance Raman spectrum of Ala45Ser Anabaena ferredoxin was distinct from the spectrum of the WT protein and showed exceptional similarity to the spectrum of higher plant ferredoxins, such as spinach ferredoxin. The mutant protein spectrum displayed considerably greater deuterium dependent isotope shifts for bands ascribed to terminal Fe-S stretching modes than did the WT spectrum. The larger shifts were attributed to a greater degree of hydrogen bonding between the protein matrix and the ligand cysteinyl sulfur atoms in the Ala45Ser variant than in the WT protein. The midpoint redox potential of Ala45Ser Anabaena ferredoxin (-382 mV vs NHE) was notably higher than that of the WT protein (-406 mV) but not in line with those of plant ferredoxins, such as the spinach protein (-420 mV). Altogether, the spectrochemical differences between the WT and Ala45Ser Anabaena ferredoxins were ascribed to the presence of an additional hydrogen bond from the side chain hydroxyl group of serine 45 to the sulfur atom of Cys 41 in the mutant protein. The distinction between the spectroscopic properties of the Ala45Ser and the WT Anabaena ferredoxins and the similarities of the spectral features of the mutant to those of higher plant ferredoxins demonstrate that the identity of the residue at position 45 tunes the microenvironment of the iron-sulfur cluster and primarily dictates the spectrochemical properties of these important proteins. Copyright 1995, American Chemical Society.

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