Kinetics of Proton Uptake and Dye Binding by Photoactive Yellow Protein in Wild Type and in the E46Q and E46A Mutants

Abstract

We studied the kinetics of proton uptake and release by photoactive yellow protein (PYP) from Ectothiorhodospira halophila in wild type and the E46Q and E46A mutants by transient absorption spectroscopy with the pH-indicator dyes bromocresol purple or cresol red in unbuffered solution. In parallel, we investigated the kinetics of chromophore protonation as monitored by the rise and decay of the blue-shifted state I(2) (lambda(max) = 355 nm). For wild type the proton uptake kinetics is synchronized with the fast phase of I(2) formation (tau = 500 micros at pH 6.2). The transient absorption signal from the dye also contains a slower component which is not due to dye deprotonation but is caused by dye binding to a hydrophobic patch that is transiently exposed in the structurally changed and partially unfolded I(2) intermediate. This conclusion is based on the wavelength, pH, and concentration dependence of the dye signal and on dye measurements in the presence of buffer. SVD analysis, moreover, indicates the presence of two components in the dye signal: protonation and dye binding. The dye binding has a rise time of about 4 ms and is coupled kinetically with a transition between two I(2) intermediates. In the mutant E46Q, which lacks the putative internal proton donor E46, the formation of I(2) is accelerated, but the proton uptake kinetics remains kinetically coupled to the fast phase of I(2) formation (tau = 100 micros at pH 6.3). For this mutant the protein conformational change, as monitored by the dye binding, occurs with about the same time constant as in wild type but with reduced amplitude. In the alkaline form of the mutant E46A the formation of the I(2)-like intermediate is even faster as is the proton uptake (tau = 20 micros at pH 8.3). No dye binding occurred in E46A, suggesting the absence of a conformational change. In all of the systems proton release is synchronized with the decay of I(2). Our results support mechanisms in which the chromophore of PYP is protonated directly from the external medium rather than by the internal donor E46.

Light-induced Proton Release of Phytochrome Is Coupled to the Transient Deprotonation of the Tetrapyrrole Chromophore

“…The dependence of the (maximal) absorbance changes on the dye concentration (Fig. 5B) allows determining the number of protons transiently released per photo-converted protein (19), yielding a value of 1.1 Ϯ 0.2. However, proton release is not fully reversed (Ͻ50%; Fig.…”

“…The signals were corrected for energy fluctuations of the pulses and irreversible bleaching, which was found to be 10% after 450 laser flashes. Photoinduced proton release (and uptake) of BV-Agp1 was determined by monitoring the absorbance changes of the pH indicator dye cresol red (pK a ϭ 8.2) at 570 nm as described (15,19). Transient absorption data at multiple wavelengths ⌬A(,t) were analyzed by singular value decomposition (SVD) and simultaneous fitting (20,21).…”

PAS Domain Allostery and Light-induced Conformational Changes in Photoactive Yellow Protein upon I2 Intermediate Formation, Probed with Enhanced Hydrogen/Deuterium Exchange Mass Spectrometry

“…In solution, additional structural and dynamic changes during I 2 formation have been observed with kinetic and thermodynamic measurements; [19][20][21] surface plasmon resonance, 22 NMR, 23,24 Fourier transform infrared (FTIR), 25,26 and CD spectroscopy; 27-30 small-angle X-ray scattering; 28,31,32 transient dye binding; 27,33,34 and computational studies. 35,36 However, the many results from X-ray crystallography and other biophysical techniques have prompted interpretations for the I 2 structure at opposite ends of the orderdisorder spectrum.…”

Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds

“…In the pR-like species, the chromophore hydrogen bonding network is either fully or partially conserved (13,14), and in the pB-like species, the pCA chromophore is protonated after breakage of the hydrogen bonding network (18). After protonation, structural changes occur in the surrounding protein (19,20) and involve unfolding of the two N-terminal helices (21,22). Although direct evidence is lacking (1), it is thought that pB is the signaling state of the protein (2,3) and that structural changes in the N-terminal helices mediate its signaling activity (3).…”