J. Meyer et al., ASSEMBLY OF A [2FE-2S](2-PASTEURIANUM RUBREDOXIN() CLUSTER IN A MOLECULAR VARIANT OF CLOSTRIDIUM), Biochemistry, 36(43), 1997, pp. 13374-13380
The rubredoxin from Clostridium pasteurianum contains a single iron at
om bound to the polypeptide chain by cysteines 6, 9, 39, and 42. The C
42A variant of this protein has been prepared by site-directed mutagen
esis and heterologous expression of the gene in Escherichia coli, The
mutated protein was found to contain an unexpected chromophore that ha
s been characterized by a variety of techniques, UV-visible absorption
and resonance Raman spectra were strongly reminiscent of those of [2F
e-2S] proteins, Mossbauer spectra of the oxidized chromophore isolated
in oxygen-free conditions indicated low-temperature diamagnetism resu
lting from antiferromagnetically coupled high-spin ferric ions. Analys
is of X-ray absorption fine structure spectra yielded an Fe-Fe distanc
e of 2.68 Angstrom, Colorimetric assays of iron and inorganic sulfide
showed that the two elements are present in a 1:1 ratio, Electrospray-
ionization mass spectra displayed a major component at M = 6190 Da, i.
e. the molecular mass of the C42A apoprotein plus two atomic masses of
iron and two atomic masses of sulfur. Taken together, these data show
that a mere point mutation allows the stabilization of a binuclear [2
Fe-2S] cluster in a protein that normally accommodates a mononuclear F
e(Scys)(4) site, Assembly of a [2Fe-2S] cluster may occur because rubr
edoxin assumes a similar fold around its metal center as the [2Fe-2S]
Rieske protein, Alternatively, a more extensive structural rearrangeme
nt of the polypeptide chain of the C42A rubredoxin variant may be cons
idered as well.