The anaerobic ribonucleotide reductase of Escherichia coli catalyzes the synthesis of the deoxyribonucleotides required for anaerobic DNA synthesis. The enzyme is an ␣ 2 ␤ 2 heterotetramer. In its active form, the large ␣ 2 subunit contains an oxygen-sensitive glycyl radical, whereas the ␤ 2 small protein harbors a

A series of gallium(III) and iron(III) complexes with five different 4N-substituted alpha-N-heterocyclic thiosemicarbazones, viz., 2-acetylpyridine N,N-dimethylthiosemicarbazone (1), 2-acetylpyridine N-pyrrolidinylthiosemicarbazone (2), acetylpyrazine N,N-dimethylthiosemicarbazone (3), acetylpyrazine N-pyrrolidinylthiosemicarbazone (4), and acetylpyrazine N-piperidinylthiosemicarbazone (5), with the general formula [GaLCl2] (HL = 1 and 2) and [ML2][Y] (M = Ga, HL = 1-5, Y = PF6; M = Fe, HL = 1-5, Y = FeCl4 and PF6) were synthesized and characterized by elemental analysis, a number of spectroscopic methods (NMR, IR, UV-vis), mass spectrometry, and X-ray crystallography. The in vitro antitumor potency was studied in two human cancer cell lines (41M and SK-BR-3). The central metal ions exert pronounced effects in a divergent manner: gallium(III) enhances, whereas iron(III) weakens the cytotoxicity of the ligands. The capacity of ligand 1 and its Ga(III) and Fe(III) complexes to destroy the tyrosyl radical of the presumed target ribonucleotide reductase is reported.

The anaerobic ribonucleoside triphosphate reductase of Escherichia coli is an iron-sulfur protein carrying an oxygen-sensitive organic radical, which is essential for catalysis. The radical was tentatively proposed to be on glycine 681, based on a comparison with the glycyl radical-containing enzyme pyruvate formate-lyase. By EPR spectroscopy of selectively 2 Hand 13 C-labeled anaerobic ribonucleotide reductase, the radical was now unambiguously assigned to carbon-2 of a glycine residue. The large 1 H hyperfine splitting (1.4 millitesla) was assigned to the ␣-proton. Site-directed mutagenesis was used to change glycine 681 into an alanine residue. In separate experiments, the two adjacent residues, cysteine 680 and tyrosine 682, were changed into serine and phenylalanine, respectively. All mutated proteins were retained on dATP-Sepharose, indicating that the mutant proteins had intact allosteric sites. They also contained amounts of iron comparable with the wild type reductase and showed the same iron-sulfur-related spectrum, suggesting that the mutant proteins were properly folded. Of the three mutant proteins only the G681A protein completely lacked the detectable glycyl radical as well as enzyme activity. Our results identify glycine 681 as the stable free radical site in E. coli anaerobic ribonucleotide reductase.

Key Points Question Which prospectively assessed descriptor of the acute pain trajectory in the first 10 days after surgery best estimates the likelihood of remote pain resolution, opioid cessation, and patient-reported complete recovery after surgery? Findings In this secondary analysis of a randomized clinical trial of 422 patients, the worst surgical-site pain intensity over the last 24 hours reported on postoperative day 10 appeared to be the best predictor of remote pain resolution, opioid cessation, and complete recovery after surgery. Meaning A possibly uniform predictor of disparate surgical outcomes long after hospital discharge may be easily assessed.