DYNAMICS OF THE N-TERMINAL ALPHA-HELIX UNFOLDING IN THE PHOTOREVERSION REACTION OF PHYTOCHROME-A

Time-resolved circular dichroism spectroscopy in the far-UV spectral r egion was used to examine the intermediates of the phytochrome photore version reaction (Pfr --> Pr). Three intermediates, lumi-F (tau = 320 ns), meta-Fa (tau = 265 mu s) and meta-Fb (tau = 5.5 ms), have been id entified in a simple sequential kinetic photoreversion mechanism by ab sorption spectroscopy [Linschitz, H., Kasche, V., Butler, W. L., & Sie gelman, H. W. (1966) J. Biol. Chem. 241, 3395-3403; Pratt, L. H., & Bu tler, Wt L. (1968) Photochem. Photobiol. 8, 477-485; Burke, M., Pratt, D. C., & Moscowitz, A. (1972) Biochemistry II, 4025-4031; Spruit, C. J. P., Kendrick, R. E., & Cooke, R. J. (1975) Planta (Berlin) 127, 121 -132; Eilfeld, P., & Rudiger, W. (1985) Z. Naturforsch. 40c, 109-114; Chen, E., Lapko, V. N., Lewis, J. W., Song, P.-S., & Kliger, D. S. (19 96) Biochemistry, 35, 843-850]. In order to correlate the unfolding of the N-terminal alpha-helical segment with one or more of the intermed iate species, time-resolved methods were coupled with the structurally sensitive probe of CD in the far-UV spectral region. Analysis of the TRCD data associates the decrease in alpha-helical content that occurs upon formation of Pr with decay of the meta-Fa intermediate. This unf olding process occurs with a time constant of 310 +/- 125 mu s, which is consistent with the 265-mu s lifetime for meta-Fa.