In Azotobacter vinelandii and Escherichia coli NifS or NifS-like
proteins are involved in FeS
protein assembly by mobilizing
sulfur from free
cysteine. This
sulfur together with Fe(2+) is then incorporated into apo-FeS
proteins to form an FeS center. A different activity termed C-DES [for
cyst(e)ine desulfurylase] was recently isolated from the cyanobacterium Synechocystis PCC 6714 which also mobilized
sulfur and which was able to incorporate the FeS center into
apoferredoxin. In the genome of the cyanobacterium Synechocystis PCC 6803, there are three open reading frames (orfs) that are similar to NifS and one that is similar to C-DES, indicating that this bacterium might contain both activities, NifS and C-DES. One orf from Synechocystis PCC 6803 encoding a NifS-like
protein, slr0387, was overexpressed in E. coli and purified. The molecular mass of the
recombinant protein was determined to be about 82 kDa, indicating that it is a homodimer. The absorption spectrum was typical for PLP-containing
proteins with an absorption maximum at 390 nm at pH 9.0 and at 425 nm at pH 6.5. The pH dependence of the absorption spectrum correlated with
enzyme activity. Maximal activity measured as
sulfide production was observed between pH 8.5 and 10. The activity decreased at lower pH values and was undetectable at pH 5.5. pH-dependent changes in the absorption spectrum and activity were attributed to protonation of the
Schiff base formed by a
lysine side chain and the PLP cofactor. Studies on substrate specificity demonstrated that
cysteine derivatives other than
cysteine methyl ester and
cysteine-sulfinic acid could not serve as substrates for this
enzyme. In particular,
cystine was not a substrate for the Synechocystis NifS-like
protein, whereas it is the best substrate for C-DES. In the presence of Fe(2+),
cysteine, and a
reductant, the NifS-like
protein was able to produce holoferredoxin from
apoferredoxin. The implications of two different activities for FeS center biosynthesis in Synechocystis are discussed.