Optical, EPR, and 1H NMR spectroscopy of serine-ligated [2Fe-2S] ferredoxins produced by site-directed mutagenesis of cysteine residues in recombinant Anabaena 7120 vegetative ferredoxin.

Abstract

Anabaena 7120 vegetative ferredoxin is a plant-type [2Fe-2S] ferredoxin that contains only four cysteine residues. The four cysteines (Cys41, Cys46, Cys49, and Cys79), 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 1H 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 (Cys46Ser, Cys49Ser, and Cys79Ser) exhibited a rhombic EPR spectrum in the reduced state, but one (Cys41Ser) showed a near-axial EPR spectrum. The 1H 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 1H NMR spectrum of the reduced Cys46Ser mutant resembled that of wild-type ferredoxin, but that of the Cys49Ser mutant did not. The hyperfine-shifted resonances of the 1H NMR spectrum of the reduced Cys46Ser mutant were assigned on the basis of results from temperature dependence studies, measurements of nuclear Overhauser effect, and 1H NMR spectra of the mutant labeled with [beta-2H]cysteine. Four hyperfine-shifted peaks of reduced Cys49Ser 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 Cys49Ser mutant showed an additional 1H 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 Cys46Ser has a "plant-type" cluster but that mutant Cys49Ser 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.(ABSTRACT TRUNCATED AT 400 WORDS)