Real-time identification of two substrate-binding intermediates for the light-driven sodium pump rhodopsin

Abstract

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Reisomerization of retinal represents a molecular switch mediating Na+ uptake and release by a bacterial sodium-pumping rhodopsin

“…The time-resolved spectra showed an ~10-nm blueshift from T = 0 to 1 s, and it then slightly redshifted afterward. Previously, we carried out the nanosecond flash photolysis of IaNaR at room temperature (7) and observed the redshifted intermediates in the millisecond region as shown in Figure 2B. The detailed kinetic analysis revealed that the millisecond process includes the four redshifted intermediates referred to as O1, O2, N, and NaR' (Figure 1C).…”

“…The sample was sealed with an O ring and another glass plate, and set in the liquid-nitrogen cooled cryostat (Optstat DN2; Oxford Instruments) for the spectroscopic measurements. mM NaCl) measured in our previous nanosecond flash photolysis study (7) at room temperature.…”

“…The spectra are plotted at selected time points-namely, 2.0 ms (red), 7.1 ms (blue), 30 ms (purple), and 135 ms (cyan)-and O1, O2, N, and NaR' make the major contributions to these four spectra, respectively (7).…”

“…However, recent studies came to find the evidences of multiple O intermediates. The X-ray crystallographic studies of a NaR from Krokinobacter eikastus (KR2), which were independently carried out by Skopintsev et al(13) and Kovalev et al(14) We recently measured the time-resolved absorption spectra of a NaR from Indibacter alkaliphilus (IaNaR), and revealed the clear presence of two O intermediates, O1 and O2, exhibiting distinct spectra and kinetics (7). The photocycle of IaNaR is shown in Figure 1C, where O1 (lifetime: 2.4 ms) has an absorption maximum (max) at 596 nm, while the subsequently-formed O2 (lifetime: 7.6 ms) has a max at 570 nm.…”

“…(C), photocycle of Ia NaR from ref. ( 14 ). The time constants shown with the photointermediates are obtained at 298 K in Tris–HCl buffer (pH 8) with 50 mM NaCl.…”