OPTICAL, EPR, AND H-1-NMR SPECTROSCOPY OF SERINE-LIGATED [2FE-2S] FERREDOXINS PRODUCED BY SITE-DIRECTED MUTAGENESIS OF CYSTEINE RESIDUES IN RECOMBINANT ANABAENA-7120 VEGETATIVE FERREDOXINS

Anabaena 7120 vegetative ferredoxin is a plant-type [2Fe-2S] ferredoxin that contains only four cysteine residues. The four cysteines (Cys(41), Cys(46), Cys(49), and Cys(79)), which ligate the iron-sulfur cluster, were mutated individually to serine. The wild-type and mutant apoprotein genes were overexpressed in Escherichia coli, and the iron-sulfur cluster was assembled in vitro by adding iron and sulfide. UV-vis, EPR, and H-1 NMR spectra were recorded on the wild-type ferredoxin and mutants. The optical spectra of all mutant proteins, in the oxidized state, differed from that of wild-type ferredoxin. Three of the mutant proteins (Cys(46)Ser, Cys(49)Ser, and Cys(79)Ser) exhibited a rhombic EPR spectrum in the reduced state, but one (Cys(41)Ser) showed a near-axial EPR spectrum. The H-1 NMR spectra of each of the four oxidized mutants contained a group of broad, hyperfine-shifted peaks between 20 and 30 ppm with anti-Curie temperature dependence. The pattern of these peaks was different for each mutant, and all were distinct from that of the wild-type ferredoxin. Because of problems with protein stability, it was possible to obtain NMR spectra of only two of the mutants when reduced. The downfield hyperfine H-1 NMR spectrum of the reduced Cys(46)Ser mutant resembled that of wild-type ferredoxin, but that of the Cys(49)Ser mutant did not. The hyperfine-shifted resonances of the H-1 NMR spectrum of the reduced Cys(46)Ser mutant were assigned on the basis of results from temperature dependence studies, measurements of nuclear Overhauser effect, and H-1 NMR spectra of the mutant labeled with [beta-H-2]cysteine. Four hyperfine-shifted peaks of reduced Cys(49)Ser at 298 K were observed at 173, 120, 32, and 18 ppm. These peaks exhibited Curie-type temperature dependence and were tentatively assigned to protons from residues coordinated to Fe(III). The reduced Cys(49)Ser mutant showed an additional H-1 NMR peak at -15 ppm (at 298 K) with Curie-type temperature dependence whose origin is unknown at present. [2Fe-2S] clusters can be placed into three different classifications according to their EPR lines shapes, NMR spectra, and reduction potentials: plant type, vertebrate type, and Rieske type. The EPR and NMR results obtained here reveal that mutant Cys(46)Ser has a ''plant-type'' cluster but that mutant Cys(49)Ser has a ''vertebrate-type'' cluster. Cysteine to serine mutations have been employed in the past to probe whether particular cysteine residues participate as iron-sulfur ligands. The present results, which show that serine can substitute for cysteine as a ligand to an iron-sulfur cluster, indicate that results of such mutations should be interpreted with caution. Nature uses cysteine exclusively in preference to serine as cluster ligands in [2Fe-2S] ferredoxins apparently on account of the higher stability.