Enzymatic regulation of the radical content of the small subunit of Escherichia coli ribonucleotide reductase involving reduction of its redox centers
- Karolinska Institutet, Stockholm (Sweden)
The active form of protein B2, a homodimeric subunit of Escherichia coli ribonucleotide reductase, contains a diferric iron center and a cationic free radical localized to tyrosine 122 of one of the two polypeptide chains. Hydroxyurea scavenges this radical but leaves the iron center intact. The resulting metB2 (earlier named B2/HU) is enzymatically inactive. Crude extracts of E. coli catalyze the interconversion of metB2 and B2. Radical introduction into metB2 requires a flavin reductase together with a second poorly defined protein fraction ( Fraction b ) as well as dioxygen, NAD(P)H, and a flavin. We now find that ferrous ions can substitute for Fraction b and that the diferric center of metB2 is reduced during anaerobic incubation of the system with reduced flavin and ferrous ions. Spectroscopic evidence and isotope experiments suggest an in situ reduction of the diferric to a diferrous center. Admission of oxygen then results in the instantaneous oxidation of tyrosine 122 to the cationic radical coupled to the reformation of the diferric center, giving enzymatically active B2. These data suggest that reduced diferrous B2 is an intermediate between metB2 and B2 during radical introduction. In addition, we find that anaerobic incubation of B2 with reduced flavin results in the loss of the tyrosyl radical and the formation of metB2. This reaction occurs in the absence of Fraction b or ferrous ions. Our experiments reconstitute with defined reagents the interconversion between metB2 and B2 observed earlier in the E. coli extract. The flavin reductase system catalyzes the interconversion in both directions with dioxygen as the critical factor deciding whether activation or inactivation of ribonucleotide reductase occurs.
- OSTI ID:
- 5564392
- Journal Information:
- Journal of Biological Chemistry; (USA), Vol. 264:16; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
IRON COMPOUNDS
REDOX REACTIONS
OXIDOREDUCTASES
CHEMICAL COMPOSITION
RADICALS
BIOSYNTHESIS
BIOCHEMICAL REACTION KINETICS
CATTLE
ESCHERICHIA COLI
FRACTIONATION
HYDROXYUREA
IRON ISOTOPES
ISOALLOXAZINES
OXYGEN
PROTEINS
REAGENTS
SPECTROPHOTOMETRY
TRACER TECHNIQUES
TYROSINE
AMIDES
AMINO ACIDS
ANIMALS
BACTERIA
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DOMESTIC ANIMALS
ELEMENTS
ENZYMES
HETEROCYCLIC COMPOUNDS
HYDROXY ACIDS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
MAMMALS
MICROORGANISMS
NONMETALS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
REACTION KINETICS
RUMINANTS
SEPARATION PROCESSES
SYNTHESIS
TRANSITION ELEMENT COMPOUNDS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques