Autocatalytic generation of Dopa in the engineered protein R2 F208Y from Escherichia coli ribonucleotide reductase and crystal structure of the Dopa-208 protein

Abstract

The mutant form Phe-208-->Tyr of the R2 protein of Escherichia coli ribonucleotide reductase contains an intrinsic ferric-Dopa cofactor with characteristic absorption bands at 460 and ca. 700 nm [Ormö, M., de Maré, F., Regnström, K., Aberg, A., Sahlin, M., Ling, J., Loehr, T. M., Sanders-Loehr, J., & Sjöberg, B. M. (1992) J. Biol. Chem. 267, 8711-8714]. The three-dimensional structure of the mutant protein, solved to 2.5-A resolution, shows that the Dopa is localized to residue 208 and that it is a bidentate ligand of Fe1 of the binuclear iron center of protein R2. Nascent apoR2 F208Y, lacking metal ions, can be purified from overproducing cells grown in iron-depleted medium. ApoR2 F208Y is rapidly and quantitatively converted to the Dopa-208 form in vitro by addition of ferrous iron in the presence of oxygen. Other metal ions (Cu2+, Mn2+, Co2+) known to bind to the metal site of wild-type apoR2 do not generate a Dopa in apoR2 F208Y. The autocatalytic generation of Dopa does not require the presence of a tyrosine residue at position 122, the tyrosine which in a wild-type R2 protein acquires the catalytically essential tyrosyl radical. It is proposed that generation of Dopa initially follows the suggested reaction mechanism for tyrosyl radical generation in the wild-type protein and involves a ferryl intermediate, which in the case of the mutant R2 protein oxygenates Tyr 208. This autocatalytic metal-mediated reaction in the engineered R2 F208Y protein may serve as a model for formation of covalently bound quinones in other proteins.

A New Tyrosyl Radical on Phe208 as Ligand to the Diiron Center in Escherichia coli Ribonucleotide Reductase, Mutant R2-Y122H

“…When R2-Y122H was isolated from cells grown in iron-free minimal medium, we obtained a similar optical spectrum without the bands at 325 and 370 nm (not shown). In consistency with previous studies of wild-type R2 from iron-free growth medium (27); however, a residual amount of up to 0.2 irons/ dimer could also be detected here using the bathophenanthroline assay.…”

“…Expression and Purification-The cells carrying the R2-Y122H mutation were grown either in Luria-Bertani (LB) medium, or in minimal medium (27). Both media contained carbenicillin 50 mg/ml and kanamycin 50 mg/ml.…”

“…The iron content in the 57 Fe solution was determined using the bathophenanthroline assay (29) after removing undissolved fragments. Cells containing the mutation R2-Y122H were grown in minimal medium as described by Åberg et al (27) except that the trace metal solution was omitted, and substituted with either 1 ml of 0.5% 56 Fe III Cl 3 ϫ 6H 2 O for the unlabeled control, or a corresponding amount of the 57 Fe stock solution for the labeled sample. This modified minimal medium was also used for global 15 N labeling and for 13 C, and 2 H isotope labeling of amino acid residues, where the medium was supplemented with sterile filtered solution of the isotope labeled L-amino acid to a final concentration of 160 M. The cells were harvested, and the protein was purified as described above.…”

Identification of an Fe(III)‐dihydroxyphenylalanine Site in Recombinant Phosphomannose Isomerase from Candida albicans

“…It was found that apo-PMI is extemely unstable and in the absence of a stabilising metal or chaotropic solution will precipitate. This prevented direct investigation of possible autocatalytic hydroxy!ation of tyrosine as seen in R2 F208Y ribonucleotide reductase (Aberg et al, 1993).…”

“…The Dopa thus formed chelates high-spin Fe(II1) giving rise to characteristic ligand to metal charge transfer bands at 460 nm and 700 nm. This reaction proceeds both in vivo (Ormo et al, 01992) and in vitro utilising purified apoprotein and iron salts (Aberg et al, 1993).…”