PH Dependence of the Photoactive Yellow Protein Photocycle Recovery Reaction Reveals a New Late Photocycle Intermediate with a Deprotonated Chromophore

Johnny Hendriks,Klaas J Hellingwerf

doi:10.1074/jbc.m805904200

Johnny Hendriks, Klaas J Hellingwerf

Open Access

https://doi.org/10.1074/jbc.m805904200

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Abstract

The recovery reaction of the signaling state of photoactive yellow protein includes the following: (i) deprotonation of the p-coumaryl chromophore, (ii) refolding of the protein, and (iii) chromophore re-isomerization from the cis to the trans configuration. Through analysis of the pH dependence of this recovery reaction, we were able to provide proof for the existence of an additional photocycle intermediate. The spectral similarity between this new intermediate and the dark state indicates that the new intermediate has a deprotonated chromophore, which may facilitate chromophore re-isomerization. This spectral similarity also explains why this new intermediate has not been noticed in earlier studies. For our data analysis we introduce a photocycle model that takes into account the effect of the specific light regime selected, a model that was also used for simulations.

Highlights

An equilibrium would result in the presence of pBЈ throughout the recovery reaction, which is not supported by this study
In this study we focus on the recovery reaction of the PYP ground state from the putative signaling state pB
In this study we have focused on the photocycle recovery reaction of PYP, i.e. the dark transition from pB to PYP ground state (pG)

Summary

EXPERIMENTAL PROCEDURES

Materials—Recombinant wild type and H108F apoPYP were produced heterologously in Escherichia coli, as described previously [26]. Sample/Buffer Preparation—Buffer solutions with defined ionic strength were prepared by mixing 1 M acetic acid, 0.2 M Na2HPO4, 1 M Trizma௡ base, 0.5 M boric acid, 4 M NaCl, 1 M HCl, and 1 M NaOH. Light Titration of H108F PYP—Transient ms/s UV/visible spectroscopy was performed on a sample containing H108F PYP in 20 mM Tris/HCl with NaCl of pH 8 and an ionic strength of 0.25 M. The sample was illuminated with varying light intensities by changing settings on the Schott KL1500 LCD lamp. The sample was illuminated until a light-induced steady state was reached. Light intensities for wavelength ranges other than 400 –700 nm were extrapolated based on the measured lamp spectra. In the time domain the data were generally analyzed from 6.5 s upward This allowed for inclusion of a small part of the light-induced steady state in the fit. The mixed spectrum (Smix) is related to the spectra for pG and pB (SpG and SpB) according to Equation 2

SpG ϩ

RESULTS

DISCUSSION

Simulation of steady state percentages of photocycle intermediates