Active-Site pKa Determination for Photoactive Yellow Protein Rationalizes Slow Ground-State Recovery

• Published in: Biophysical journal 2017-05, Vol.112 (10), p.2109-2116
• Main Authors: Oktaviani, Nur Alia, Pool, Trijntje J., Yoshimura, Yuichi, Kamikubo, Hironari, Scheek, Ruud M., Kataoka, Mikio, Mulder, Frans A.A.
• Format: Article
• Language: English
• Subjects: Aspartic Acid - chemistry; Aspartic Acid - metabolism; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Coumaric acid; Experiments; Glutamic Acid - chemistry; Glutamic Acid - metabolism; Halorhodospira halophila; Hydrogen Bonding; Hydrogen-Ion Concentration; Kinetics; Light effects; Nuclear Magnetic Resonance, Biomolecular; Optical properties; pH effects; Photoactive yellow protein; Photoreceptors, Microbial - chemistry; Photoreceptors, Microbial - metabolism; Proteins; Protonation; Protons; Recovery; Residues; Yellow protein
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2017.04.008

The ability to avoid blue-light radiation is crucial for bacteria to survive. In Halorhodospira halophila, the putative receptor for this response is known as photoactive yellow protein (PYP). Its response to blue light is mediated by changes in the optical properties of the chromophore para-coumaric acid (pCA) in the protein active site. PYP displays photocycle kinetics with a strong pH dependence for ground-state recovery, which has remained enigmatic. To resolve this problem, a comprehensive pKa determination of the active-site residues of PYP is required. Herein, we show that Glu-46 stays protonated from pH 3.4 to pH 11.4 in the ground (pG) state. This conclusion is supported by the observed hydrogen-bonded protons between Glu-46 and pCA and Tyr-42 and pCA, which are persistent over the entire pH range. Our experimental results show that none of the active-site residues of PYP undergo pH-induced changes in the pG state. Ineluctably, the pH dependence of pG recovery is linked to conformational change that is dependent upon the population of the relevant protonation state of Glu-46 and the pCA chromophore in the excited state, collaterally explaining why pG recovery is slow.