RECOMBINANT DESULFOVIBRIO-VULGARIS RUBRERYTHRIN - ISOLATION AND CHARACTERIZATION OF THE DIIRON DOMAIN

The gene encoding Desulfovibrio (D.) vulgaris rubrerythrin (Prickril, B. C,, Kurtz, D. M., Jr., LeGall, J., and Voordouw, G. (1991) Biochemistry 30, 1118), a protein of unknown function containing both FeS4 and (mu-oxo)diiron sites, was cloned and overexpressed in Escherichia coli. Upon cell lysis, the overexpressed protein was found in an insoluble form deficient in iron. Iron was incorporated in vitro by dissolving the protein in 3 M guanidinium chloride, adding Fe(II) anaerobically and diluting the denaturant. This recombinant rubrerythrin was found to have properties very similar to those of rubrerythrin isolated from D. vulgaris, except that the recombinant rubrerythrin contained six rather than four (or five) iron atoms per 44 kDa homodimer. Analyses of UV-vis, Mossbauer, and EPR spectra showed that the six iron atoms in recombinant rubrerythrin are organized as two FeS4 and two (mu-oxo/hydroxo)diiron sites. In order to allow examination of the diiron sites in the absence of the FeS4 sites, a truncated gene encoding the N-terminal 152 residues of D. vulgaris rubrerythrin was also cloned and overexpressed as an insoluble protein in E. coli, and iron was incorporated by a procedure analogous to that for recombinant rubrerythrin. This so-called ''chopped'' rubrerythrin (CRr) was found to consist of an approximate to 35 kDa homodimer containing four iron atoms. Spectroscopic characterization indicated that the four iron atoms in CRr are organized as two diiron sites, the majority of which closely resemble the (mu-oxo)diiron(III) sites in E. coli ribonucleotide reductase R2 protein, and a minor fraction of which resemble the mixed-valent diiron(II,III) site in methane monooxygenase hydroxylase. Both the diiron(III) and diiron(II) sites of CRr were shown by UV-vis and Mossbauer spectroscopies to form complexes with azide. The diiron(II)CRr azide adduct could be detected by a parallel-mode EPR signal at g approximate to 17.5. A similar parallel-mode EPR signal at g approximate to 19.8 was detected when excess azide was added to the reduced recombinant rubrerythrin. EPR-based redox titrations of CRr gave diiron(III)-to-mixed-valent and mixed-valent-to-diiron(II) reduction potentials of 215 +/- 5 and 154 +/- 5 mV, respectively, vs NHE. These potentials were approximately 70 mV more negative than those measured for the corresponding redox couples in recombinant rubrerythrin. Possible activities for rubrerythrin are discussed.