The environment of [2Fe-2S] clusters in ferredoxins: the role of residue 45 probed by site-directed mutagenesis.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Vidakovic
M
M
Department of Chemistry, University of Houston, Texas 77204-5641, USA.
Fraczkiewicz
G
G
Dave
B C
BC
Czernuszewicz
R S
RS
Germanas
J P
JP
D017033:Anabaena / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
D001426:Bacterial Proteins; D017931:DNA Primers; D005288:Ferredoxins; D011994:Recombinant Proteins; C064537:petF protein, Anabaena