Spectrally silent transitions in the bacteriorhodopsin photocycle

Abstract

The photocycle kinetics of bacteriorhodopsin were analyzed from 0 to 40 degrees C at 101 wavelengths (330-730 nm). The data can be satisfactorily approximated by eight exponents. The slowest component (half-time 20 ms at 20 degrees C) belongs to the 13-cis cycle. The residual seven exponentials that are sufficient to describe the all-trans photocycle indicate that at least seven intermediates of the all-trans cycle must exist, although only five spectrally distinct species (K, L, M, N, and O) have been identified. These seven exponentials and their spectra at different temperatures provide the basis for the discussion of various kinetic schemes of the relaxation. The simplest model of irreversible sequential transitions includes after the first K--> L step the quasiequilibria of L<-->M, M<-->N, and N<-->O intermediates. These quasiequilibria are controlled by rate-limiting dynamics of the protein and/or proton transfer steps outside the chromophore region. Thus there exists an apparent kinetic paradox (i.e., why is the number of exponents of relaxation (at least seven) higher than the number of distinct spectral intermediates (only five)), which can be explained by assuming that some of the transitions correspond to changes in the quasiequilibria between spectrally distinct intermediates (i.e., are spectrally silent).

Water Structural Changes in the L and M Photocycle Intermediates of Bacteriorhodopsin as Revealed by Time-Resolved Step-Scan Fourier Transform Infrared (FTIR) Spectroscopy

“…The contributions of the photocycle intermediates to the b-spectra in (32). The rest (70%) should be due to N. This spectrum is identical to that obtained by exchanging the order of the last two time constants (k 5 and k 6 , in our case) as proposed by Chizhov et al (43), and similar to the penultimate spectrum shown in Figure 3A of Rödig et al (28). Spectrum b 6 (h) is roughly similar to b 5 (f) in shape, but with only one sixth the amplitude.…”

“…The spectra obtained in this way, together with the rate constants, were then used to calculate the spectra for a reaction consisting of a linear sequence of intermediates, using the method of Chizhov et al (43).…”

Simultaneous measurements of fast optical and proton current kinetics in the bacteriorhodopsin photocycle using an enhanced spectrophotometer

“…Table 2 shows the presence of 6 kinetically distinct species with taus near 0.008, 0.05, 0.15, 0.5, 1.9, and 4.9 ms. These are essentially the same components as obtained previously by Hendler et al [14,20] and Chizhov et al [21] using optical data, and by Rodig et al [22] and Morgan et al [23 ] using FTIR data, and by Müller et al using both optical and FTIR data [24]. It is also shown in Table 2 that the component seen optically with a tau near 1.9 ms is absent in the electrical data.…”

Structural kinetics of myosin by transient time-resolved FRET

“…Transitions among distinct structural states, each characterized by both structural order and internal dynamic disorder, are typically required for the function of a protein, especially an enzyme. To describe the mechanism of a protein's function is to describe its structural kinetics, i.e., the coupling of protein structural transitions to biochemical kinetics, as defined by changes in bound ligand (1)(2)(3). However, for most proteins there remains mechanistic ambiguity due to a gap between structural data, determined primarily from static protein crystals, and kinetics, measured during the transient phase of the biochemical reaction.…”

“…All these data suggest This article is a PNAS Direct Submission. 1 To whom correspondence may be addressed. Email: yuri.nesmelov@uncc.edu or ddt@ umn.edu.…”