Abstract
Chromophore oxidation using tetranitromethane was performed with 124 kDa, 59kDa, and 39kDa oat phytochrome in both photoreversible forms (Pr and Pfr). Kinetics and intermediates of oxidation were analyzed using a diode array spectrophotometer. A transient species (λmax = 580 and 505 nm) was observed exclusively from the Pfr (or Pbl in the case of 39 kDa phytochrome) forms of degraded phytochromes. This oxidation product was isolated by rapid size-exclusion HPLC. Comparison of its absorption spectrum with those of model compounds revealed a strong similarity to biladienes-a,b, indicating that the primary attack of tetranitromethane takes place at the methine bridge, predominantly at C-15. This is interpreted in terms of a different accessibility of carbon-15 of the chromophore in the Pr vs. Pfr form.
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