Addition of oxygen to the diiron(II/II) cluster is the slowest step in formation of the tyrosyl radical in the W103Y variant of ribonucleotide reductase protein R2 from mouse.

Activation of O2 by the diiron(II/II) cluster in protein R2 of class I ribonucleotide reductase generates the enzyme's essential tyrosyl radical. A crucial step in this reaction is the transfer of an electron from solution to a diiron(II/II)-O2 adduct during formation of the radical-generating, diiron(III/IV) intermediate X. In the reaction of R2 from Escherichia coli, this electron injection is initiated by the rapid (>400 s-1 at 5 degrees C), transient oxidation of the near-surface residue, tryptophan 48, to a cation radical and is blocked by substitution of W48 with F, A, G, Y, L, or Q. By contrast, a study of the cognate reaction in protein R2 from mouse suggested that electron injection might be the slowest step in generation of its tyrosyl radical, Y177* [Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gräslund, A. (1998) J. Biol. Chem. 273, 21463-21472]. The crucial evidence was the observation that Y177* production is slowed by approximately 30-fold upon substitution of W103, the cognate of the electron-shuttling W48 in E. coli R2, with tyrosine. In this work, we have applied stopped-flow absorption and freeze-quench electron paramagnetic resonance and Mössbauer spectroscopies to the mouse R2 reaction to evaluate the possibility that an already sluggish electron-transfer step is slowed by 30-fold by substitution of this key residue. The drastically reduced accumulation of cluster X, failure of precursors to the intermediate to accumulate, and, most importantly, first-order dependence of the rate of Y177* formation on the concentration of O2 prove that addition of O2 to the diiron(II/II) cluster, rather than electron injection, is the slowest step in the R2-W103Y reaction. This finding indicates that the basis for the slowing of Y177* formation by the W103Y substitution is an unexpected secondary effect on the structure or dynamics of the protein, its diiron(II/II) cluster, or both rather than the expected chemical effect on the electron injection step.

(Mu-1,2-peroxo)diiron(III/III) complex as a precursor to the diiron(III/IV) intermediate X in the assembly of the iron-radical cofactor of ribonucleotide reductase from mouse.

Stopped-flow absorption and freeze-quench electron paramagnetic resonance (EPR) and Mössbauer spectroscopies have been used to obtain evidence for the intermediacy of a (mu-1,2-peroxo)diiron(III/III) complex on the pathway to the tyrosyl radical and (mu-oxo)diiron(III/III) cluster during assembly of the essential cofactor in the R2 subunit of ribonucleotide reductase from mouse. The complex accumulates to approximately 0.4 equiv in the first few milliseconds of the reaction and decays concomitantly with accumulation of the previously detected diiron(III/IV) cluster, X, which generates the tyrosyl radical and product (mu-oxo)diiron(III/III) cluster. Kinetic complexities in the reaction suggest the existence of an anti-cooperative interaction of the monomers of the R2 homodimer in Fe(II) binding and perhaps O2 activation. The detection of the (mu-1,2-peroxo)diiron(III/III) complex, which has spectroscopic properties similar to those of complexes previously characterized in the reactions of soluble methane monooxygenase, stearoyl acyl carrier protein Delta9 desaturase, and variants of Escherichia coli R2 with the iron ligand substitution, D84E, provides support for the hypothesis that the reactions of the diiron-carboxylate oxidases and oxygenases commence with the formation of this common intermediate.

Isolated inflammatory sphenoid sinus disease: a revisitation of computed tomography indications based on presenting findings.

BACKGROUND: Isolated inflammatory sphenoid sinus disease (IISSD) can be difficult to diagnose. Frequently, history and physical are inadequate in establishing a diagnosis. Computed tomography (CT) is an excellent screening tool; however, it often is obtained late in the disease process because of vague symptoms at presentation. Identifying the most common presenting symptoms of IISSD may allow earlier detection and avoidance of more severe sequelae by determining earlier indications for CT. Presently, headache is not an indication for sinus CT. METHODS: A retrospective chart review of IISSD presentation was performed at our institution. A literature review was performed also to quantitatively document trends in presentation of IISSD, including characterization of headache symptoms by location. Cumulative findings were then compared with current CT indications to determine if presentation patterns warrant a change in indications for CT. RESULTS: A total of 361 cases were evaluated by our inclusion criteria. Headache was the most common finding (81.7%), particularly peri/retro-orbital, vertex, and frontal headache. Ocular changes (17.5%) and cranial nerve involvement (16.1%) were common also, but headache frequently was a solitary finding (42.6%). Twenty-six IISSD cases were reviewed at our institution over 7 years, with similar results. Under current guidelines, the only IISSD findings that are indications for CT scan are the ophthalmologic and neurological complications. CONCLUSION: Not every headache necessitates a CT scan. However, the deep-seated vertex, frontal, and, particularly, peri/retro-orbital headaches, especially when aggravated by head movement and refractory to analgesics, as is often seen in IISSD, should be an indication for CT evaluation.