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)

Resonance Raman spectroscopic evidence for the FeS4 and Fe-O-Fe sites in rubrerythrin from Desulfovibrio vulgaris.

Resonance Raman (RR) spectra of the non-heme iron protein rubrerythrin from Desulfovibrio vulgaris unequivocally demonstrate the presence of both a rubredoxin-type FeS4 site and a (mu-oxo)diiron(III) cluster. The RR spectra of rubrerythrin excited at 496.5 and 568.2 nm are dominated by bands similar to those of rubredoxin and conform to the vibrational pattern expected for a distorted FeS4 tetrahedron of an Fe(S-Cys)4 site. Numerous overtone and combination bands of the Fe-S stretches are also observed, and a band at 650 cm-1 is assigned to a cysteine C-S stretching mode. The 374-, 355-, and 340-cm-1 bands, assigned to the three components of the v3(T2) asymmetric FeS4 stretching mode, are 2-8 cm-1 lower than the corresponding frequencies for the Desulfovibrio gigas rubredoxin FeS4 site. Similar differences in frequencies of bands assigned to SFeS bending modes between rubredoxin and rubrerythrin are also detected. These frequency differences imply either slightly weaker Fe-S bonds or subtle conformational differences among the cysteinyl ligands in the rubrerythrin versus rubredoxin FeS4 sites. The RR spectrum of rubrerythrin excited at 406.7 nm shows dramatically diminished intensities of the FeS4 bands with concomitant enhancement of a band at 514 cm-1. This band shifts 18 cm-1 to lower frequency when the protein is dissolved in H(2)18O. The frequency of this band and the 18O isotope shift are those expected for the symmetric Fe-O-Fe stretch of a bent oxo-bridged diiron(III) cluster and indicate that this cluster has at least one additional bridging ligand.(ABSTRACT TRUNCATED AT 250 WORDS)