Publications

The reaction of oxidized bovine cytochrome c oxidase (bCcO) with hydrogen peroxide (H(2)O(2)) was studied by electron paramagnetic resonance (EPR) to determine the properties of radical intermediates. Two distinct radicals with widths of 12 and 46 G are directly observed by X-band EPR in the reaction of bCcO with H(2)O(2) at pH 6 and pH 8. High-frequency EPR (D-band) provides assignments to tyrosine for both radicals based on well-resolved g-tensors. The wide radical (46 G) exhibits g-values similar to a radical generated on L-Tyr by UV-irradiation and to tyrosyl radicals identified in many other enzyme systems. In contrast, the g-values of the narrow radical (12 G) deviate from L-Tyr in a trend akin to the radicals on tyrosines with substitutions at the ortho position. X-band EPR demonstrates that the two tyrosyl radicals differ in the orientation of their β-methylene protons. The 12 G wide radical has minimal hyperfine structure and can be fit using parameters unique to the post-translationally modified Y244 in bCcO. The 46 G wide radical has extensive hyperfine structure and can be fit with parameters consistent with Y129. The results are supported by mixed quantum mechanics and molecular mechanics calculations. In addition to providing spectroscopic evidence of a radical formed on the post-translationally modified tyrosine in CcO, this study resolves the much debated controversy of whether the wide radical seen at low pH in the bovine enzyme is a tyrosine or tryptophan. The possible role of radical formation and migration in proton translocation is discussed.

The vertebral column derives from somites, which are transient paired segments of mesoderm that surround the neural tube in the early embryo. Somites are formed by a genetic mechanism that is regulated by cyclical expression of genes in the Notch, Wnt, and fibroblast growth factor (FGF) signaling pathways. These oscillators together with signaling gradients within the presomitic mesoderm help to set somitic boundaries and rostral-caudal polarity that are essential for the precise patterning of the vertebral column. Disruption of this mechanism has been identified as the cause of severe segmentation defects of the vertebrae in humans. These segmentation defects are part of a spectrum of spinal disorders affecting the skeletal elements and musculature of the spine, resulting in curvatures such as scoliosis, kyphosis, and lordosis. While the etiology of most disorders with spinal curvatures is still unknown, genetic and developmental studies of somitogenesis and patterning of the axial skeleton and musculature are yielding insights into the causes of these diseases.