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 t he [2Fe-2S] ferredoxin from vegetative cells of the cyanobacterium Ana baena sp. 7120 are described. This novel protein, which incorporates t he residue present in many higher plant ferredoxins into the analogous position of a typical cyanobacterial ferredoxin, was prepared to prob e the origin of the characteristic spectrochemical and functional diff erences between the ferredoxins from these two sources. The variant pr otein was produced by site-directed mutagenesis and was expressed as t he holoprotein in Escherichia coli. Although the UV-vis spectrum of th e Ala45Ser mutant was indistinguishable from that of the wild-type (WT ) protein, the circular dichroism (CD) spectrum of the mutant was dist inct and similar in appearance to that of spinach ferredoxin, which po ssesses a Ser residue at the analogous position. The values of the pri ncipal 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 mu tant EPR spectrum according to the method of Blumberg indicated greate r covalent interactions between the localized ferrous site of the clus ter and the protein matrix relative to the WT protein, The resonance R aman spectrum of Ala45Ser Anabaena ferredoxin was distinct from the sp ectrum of the WT protein and showed exceptional similarity to the spec trum of higher plant ferredoxins, such as spinach ferredoxin. The muta nt protein spectrum displayed considerably greater deuterium dependent isotope shifts for bands ascribed to terminal Fe-S stretching modes t han did the WT spectrum, The larger shifts were attributed to a greate r 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 m V vs NHE) was notably higher than that of the WT protein (-406 mV) bur nor in line with those of plant ferredoxins, such as the spinach prot ein (-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 ser ine 45 to the sulful atom of Cys 41 in the mutant protein, The distinc tion between the spectroscopic properties of the Ala45Ser and the WT A nabana ferredoxins and the similarities of the spectral features of th e mutant to those of higher plant ferredoxins demonstrate that the ide ntity of the residue at position 45 tunes the microenvironment of the iron-sulfur cluster and primarily dictates the spectrochemical propert ies of these important proteins.