Two-photon Absorption and Photoionization of a Bacterial Phytochrome

Thanh Nhut Do; David Menendez; Dorina Bizhga; Emina A Stojković; John T M Kennis

doi:10.1016/j.jmb.2023.168357

Two-photon Absorption and Photoionization of a Bacterial Phytochrome

J Mol Biol. 2024 Mar 1;436(5):168357. doi: 10.1016/j.jmb.2023.168357. Epub 2023 Nov 7.

Authors

Thanh Nhut Do¹, David Menendez², Dorina Bizhga², Emina A Stojković², John T M Kennis³

Affiliations

¹ Department of Physics and Astronomy, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands.
² Department of Biology, Northeastern Illinois University, 5500 N. St. Louis Ave., Chicago, IL 60625, USA.
³ Department of Physics and Astronomy, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands. Electronic address: j.t.m.kennis@vu.nl.

PMID: 37944794
DOI: 10.1016/j.jmb.2023.168357

Abstract

Phytochromes constitute a family of photosensory proteins that are utilized by various organisms to regulate several physiological processes. Phytochromes bind a bilin pigment that switches its isomeric state upon absorption of red or far-red photons, resulting in protein conformational changes that are sensed by the organism. Previously, the ultrafast dynamics in bacterial phytochrome was resolved to atomic resolution by time-resolved serial femtosecond X-ray diffraction (TR-SFX), showing extensive changes in its molecular conformation at 1 picosecond delay time. However, the large excitation fluence of mJ/mm² used in TR-SFX questions the validity of the observed dynamics. In this work, we present an excitation-dependent ultrafast transient absorption study to test the response of a related bacterial phytochrome to excitation fluence. We observe excitation power-dependent sub-picosecond dynamics, assigned to the population of high-lying excited state S_n through resonantly enhanced two-photon absorption, followed by rapid internal conversion to the low-lying S₁ state. Inspection of the long-lived spectrum under high fluence shows that in addition to the primary intermediate Lumi-R, spectroscopic signatures of solvated electrons and ionized chromophore radicals are observed. Supported by numerical modelling, we propose that under excitation fluences of tens of μJ/mm² and higher, bacterial phytochrome partly undergoes photoionization from the S_n state in competition with internal conversion to the S₁ state in 300 fs. We suggest that the extensive structural changes of related, shorter bacterial phytochrome, lacking the PHY domain, resolved from TR-SFX may have been affected by the ionized species. We propose approaches to minimize the two-photon absorption process by tuning the excitation spectrum away from the S₁ absorption or using phytochromes exhibiting minimized or shifted S₁ absorption.

Keywords: biliverdin; phytochrome; power titration; solvated electron; transient absorption spectroscopy.

MeSH terms

Absorption, Physicochemical
Bacterial Proteins* / chemistry
Bile Pigments / chemistry
Isomerism
Phytochrome* / chemistry
Protein Conformation
Spectrum Analysis
X-Ray Diffraction

Substances

Bacterial Proteins
Bile Pigments
Phytochrome