Two Bathointermediates of the Bacteriorhodopsin Photocycle, from Time-Resolved Nanosecond Spectra in the Visible

Abstract

Time-resolved measurements were performed on wild-type bacteriorhodopsin with an optical multi-channel analyzer in the spectral range of 350–735 nm, from 100 ns to the photocycle completion, at four temperatures in the 5–30 °C range. The intent was to examine the possibility of two K-like bathochromic intermediates and to obtain their spectra and kinetics in the visible. The existence of a second K-like intermediate, termed KL, had been postulated (Shichida et al., BBA 723:240–246, 1983) to reconcile inconsistencies in data in the pico- and microsecond time-domains. However, introduction of KL led to a controversy since neither its visible spectrum nor its kinetics could be confirmed. Infra-red data (Dioumaev & Braiman, J. Phys. Chem., B, 101:1655–1662, 1997), revealed a state, which might have been considered a homologue to KL but it had a kinetic pattern different from that of the earlier proposed KL. Here we characterize two distinct K-like intermediates, KE (“early”) and KL (“late”), by their spectra and kinetics in the visible as revealed by global kinetic analysis. The KE-to-KL transition has a time constant of ~250 ns at 20 °C, and describes a shift from KE with λmax at ~600 nm and extinction of ~56,000 M−1·cm−1 to KL with λmax at ~590 nm and extinction of ~50,000 M−1·cm−1. The temperature dependence of this transition is characterized by an enthalpy of activation ΔH# ~ 40 kJ/mol, and a positive entropy of activation ΔS#/R = ~4. The consequences of multiple K-like states for interpreting the spectral evolution in the early stages of the photocycle are discussed.

Photocycle of Exiguobacterium sibiricum Rhodopsin Characterized by Low-Temperature Trapping in the IR and Time-Resolved Studies in the Visible

“…To obtain time-resolved spectra of intermediates, single-wavelength kinetic traces were collected separately at 18 wavelengths covering the 380–720 nm region with steps of 20 nm, and subjected to analysis using the FitExp and Schemefit programs 56 . At first global (multi-exponential) fitting was performed with FitExp program, and the minimal sufficient number of exponential components was evaluated by F-test statistics and analysis of residuals, see 56 for details.…”

“…At first global (multi-exponential) fitting was performed with FitExp program, and the minimal sufficient number of exponential components was evaluated by F-test statistics and analysis of residuals, see 56 for details. This number is equal to the number of distinct intermediates detected experimentally 56 .…”

“…At first global (multi-exponential) fitting was performed with FitExp program, and the minimal sufficient number of exponential components was evaluated by F-test statistics and analysis of residuals, see 56 for details. This number is equal to the number of distinct intermediates detected experimentally 56 . The corresponding exponentials are the apparent processes, and the difference spectra of the mixtures formed between them were calculated by SchemeFit program 56 .…”

“…This number is equal to the number of distinct intermediates detected experimentally 56 . The corresponding exponentials are the apparent processes, and the difference spectra of the mixtures formed between them were calculated by SchemeFit program 56 . These spectra are conceptually similar to that of the so-called kinetic states 57 , but include additional corrections obligatory for the real-case kinetics, in which the neighboring time constants, as is the case in ESR, are of the same order of magnitude.…”

“…In the more realistic case of a sequence with back reactions, A↔B↔C↔D↔E↔, as is the case for the ESR photocycle (see details in the Results section), the apparent rates do not correspond to simple transitions such as A-to-B and B-to-C but rather to transitions from an A state into a mixture of A and B, and later on into a more complex mixture of A and B and C states coexisting temporarily due to transient equilibria. The spectra calculated in this manner 17,56,58 are weighted sums of the pure spectra of the individual intermediates in the mixtures formed in these transient equilibria.…”