The active form of the R2F protein of class Ib ribonucleotide reductase from Corynebacterium ammoniagenes is a diferric protein.
Corynebacterium ammoniagenes contains a ribonucleotide reductase (RNR) of the class Ib type. The small subunit (R2F) of the enzyme has been proposed to contain a manganese center instead of the dinuclear iron center, which in other class I RNRs is adjacent to the essential tyrosyl radical. The nrdF gene of C. ammoniagenes, coding for the R2F component, was cloned in an inducible Escherichia coli expression vector and overproduced under three different conditions: in manganese-supplemented medium, in iron-supplemented medium, and in medium without addition of metal ions. A prominent typical tyrosyl radical EPR signal was observed in cells grown in rich medium. Iron-supplemented medium enhanced the amount of tyrosyl radical, whereas cells grown in manganese-supplemented medium had no such radical. In highly purified R2F protein, enzyme activity was found to correlate with tyrosyl radical content, which in turn correlated with iron content. Similar results were obtained for the R2F protein of Salmonella typhimurium class Ib RNR. The UV-visible spectrum of the C. ammoniagenes R2F radical has a sharp 408-nm band. Its EPR signal at g = 2.005 is identical to the signal of S. typhimurium R2F and has a doublet with a splitting of 0.9 millitesla (mT), with additional hyperfine splittings of 0.7 mT. According to X-band EPR at 77-95 K, the inactive manganese form of the C. ammoniagenes R2F has a coupled dinuclear Mn(II) center. Different attempts to chemically oxidize Mn-R2F showed no relation between oxidized manganese and tyrosyl radical formation. Collectively, these results demonstrate that enzymatically active C. ammoniagenes RNR is a generic class Ib enzyme, with a tyrosyl radical and a diferric metal cofactor.
Huque
Y
Y
Department of Molecular Biology, Arrhenius Laboratories, Stockholm University, Sweden.
Fieschi
F
F
Torrents
E
E
Gibert
I
I
Eliasson
R
R
Reichard
P
P
Sahlin
M
M
Sjoberg
B M
BM
D001426:Bacterial Proteins; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007501:Iron / Q000737:chemistry* / Q000494:pharmacology; D008024:Ligands; D008345:Manganese / Q000737:chemistry / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics*; D012486:Salmonella typhimurium / Q000201:enzymology; D013053:Spectrophotometry; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D008024:Ligands; D008345:Manganese; D007501:Iron; C114872:NrdF protein, bacteria; D012264:Ribonucleotide Reductases