Restoring proper radical generation by azide binding to the iron site of the E238A mutant R2 protein of ribonucleotide reductase from Escherichia coli.

The enzyme activity of Escherichia coli ribonucleotide reductase requires the presence of a stable tyrosyl free radical and diiron center in its smaller R2 component. The iron/radical site is formed in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. The reaction is known to proceed via a paramagnetic intermediate X, formally a Fe(III)-Fe(IV) state. We have used 9.6 GHz and 285 GHz EPR to investigate intermediates in the reconstitution reaction in the iron ligand mutant R2 E238A with or without azide, formate, or acetate present. Paramagnetic intermediates, i.e. a long-living X-like intermediate and a transient tyrosyl radical, were observed only with azide and under none of the other conditions. A crystal structure of the mutant protein R2 E238A/Y122F with a diferrous iron site complexed with azide was determined. Azide was found to be a bridging ligand and the absent Glu-238 ligand was compensated for by azide and an extra coordination from Glu-204. A general scheme for the reconstitution reaction is presented based on EPR and structure results. This indicates that tyrosyl radical generation requires a specific ligand coordination with 4-coordinate Fe1 and 6-coordinate Fe2 after oxygen binding to the diferrous site.

Crystal structures of two self-hydroxylating ribonucleotide reductase protein R2 mutants: structural basis for the oxygen-insertion step of hydroxylation reactions catalyzed by diiron proteins.

The R2 protein of ribonucleotide reductase catalyzes the dioxygen-dependent one-electron oxidation of Tyr122 at a diiron-carboxylate site. Methane monooxygenase and related hydroxylases catalyze hydrocarbon hydroxylation at diiron sites structurally related to the one in R2. In protein R2, the likely reaction site for dioxygen is close to Phe208. The crystal structure of an iron ligand mutant R2, Y122F/E238A, reveals the hydroxylation of Phe208 at the meta, or epsilon-, ring position and the subsequent coordination of this residue to the diiron site. In another mutant, F208Y, the "foreign" residue Tyr208 is hydroxylated to Dopa. The structures of apo and diferrous F208Y presented here suggest that Tyr208 is coordinated to the iron site of F208Y throughout the Dopa generation cycle. Together, the structural data on these two mutants suggest two possible reaction geometries for the hydroxylation reaction catalyzed by these modified R2 diiron sites, geometries which might be relevant for the hydroxylation reaction catalyzed by other diiron sites such as methane monooxygenase. A critical role for residue Glu238 in directing the oxidative power of the reactive intermediate toward oxidation of Tyr122 is proposed.

Longitudinal changes in hearing ability among Swedish conscripts.

An analysis of a military database of about 36,000 tone audiograms from male Swedish conscripts aged 18 to 19 and recorded from 1969 to 1977 demonstrates a successively decreasing prevalence of hearing loss during this period. This might reflect improved therapy during the 1950s and 1960s of ear disorders causing hearing loss in small children. If observations in other studies on a reverse trend during the 1980s are confirmed, they indicate, together with the present study, that around 1980 young people began to be harmfully exposed to an environmental factor causing hearing loss. If this is the case, the causative factor would probably be non-occupational exposure to electronically amplified sounds from loudspeakers and headphones.