Local electric field changes during thephotoconversion of the bathy phytochrome Agp2 [original]

Anastasia Kraskov, Johannes von Sass, Anh Duc Nguyen, Tu Oanh

Hoang, David Buhrke, Sagie Katz, Norbert Michael, Jacek Kozuch,

Ingo Zebger, Friedrich Siebert, Patrick Scheerer, Maria Andrea

Mroginski, Nediljko Budisa, Peter Hildebrandt

Local electric field changes during the

photoconversion of the bathy phytochrome Agp2

Open Access via institutional repository of Technische Universität Berlin

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Journal article | Accepted version

(i. e. final author-created version that incorporates referee comments and is the version accepted for

publication; also known as: Author’s Accepted Manuscript (AAM), Final Draft, Postprint)

This version is available at

https://doi.org/10.14279/depositonce-12714

Citation details

Kraskov, A., von Sass, J., Nguyen, A. D., Hoang, T. O., Buhrke, D., Katz, S., Michael, N., Kozuch, J., Zebger, I.,

Siebert, F., Scheerer, P., Mroginski, M. A., Budisa, N., Hildebrandt, P. (2021). Local Electric Field Changes

during the Photoconversion of the Bathy Phytochrome Agp2. In Biochemistry (Vol. 60, Issue 40, pp.

2967–2977). American Chemical Society (ACS). https://doi.org/10.1021/acs.biochem.1c00426.

This document is the Accepted Manuscript version of a Published Work that appeared in final form in

To access the final edited and published work see https://doi.org/10.1021/acs.biochem.1c00426.

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Local electric field changes during the photoconversion of the bathy

phytochrome Agp2

Anastasia Kraskov§1, Johannes von Sass§1, Duc A. Nguyen1, Tu Oanh Hoang1, David Buhrke1,6,

Sagie Katz1, Norbert Michael1, Jacek Kozuch3, Ingo Zebger1, Friedrich Siebert2, Patrick

Scheerer4, Maria Andrea Mroginski1*, Nediljko Budisa5*, Peter Hildebrandt1*

1 Technische Universität Berlin, Institut für Chemie, Sekr. PC14, Straße des 17. Juni 135, D-

10623 Berlin, Germany

2 Albert-Ludwigs-Universität Freiburg, Institut für Molekulare Medizin und Zellforschung,

Sektion Biophysik, Hermann-Herderstr. 9, D-79104 Freiburg, Germany

3 Freie Universität Berlin, Fachbereich für Physik, Arnimallee 14, D-14195 Berlin, Germany

4 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and

Humboldt-Universität zu Berlin, Institute of Medical Physics and Biophysics, Group Protein

X-ray Crystallography and Signal Transduction, Charitéplatz 1, D-10117 Berlin, Germany

5 University of Manitoba, Department of Chemistry, 144 Dysart Rd, 360 Parker Building, R3T

2N2 Winnipeg, MB, Canada

6 present address: University of Zürich, Department of Chemistry, Winterthurerstr. 190, CH-

8057 Zürich, Switzerland

§ equal contributions to the work

* corresponding authors

Peter Hildebrandt: email: [email protected]

Nediljko Budisa: email: [email protected]

Maria Andrea Mroginski: email: [email protected]

Abstract

Phytochromes switch between a physiologically inactive and active state via a light-induced

reaction cascade, which is initiated by isomerization of the tetrapyrrole chromophore and leads

to the functionally relevant secondary structure transition of a protein segment (tongue).

Although details of the underlying cause-effect relationships are not known, electrostatic fields

are likely to play a crucial role in coupling chromophore and protein structural changes. Here,

we studied local electric field changes during the photoconversion of the dark state Pfr to the

photoactivated state Pr of bathy phytochrome Agp2. Substituting Tyr165 and Phe192 in the

chromophore pocket by para-cyanophenylalanine (pCNF), we monitored the respective nitrile

stretching modes in the various states of the photoconversion (vibrational Stark effect).

Resonance Raman and IR spectroscopic analyses revealed that both pCNF-substituted variants

undergo the same photoinduced structural changes as wild-type Agp2. Based on a structural

model for the Pfr state of F192pCNF, a molecular mechanical – quantum mechanical approach

was employed to calculate the electric field at the nitrile group and the respective stretching

frequency in excellent agreement with the experiment. These calculations serve as a reference

for determining the electric field changes in the photoinduced states of F192pCNF. Unlike

F192pCNF, the nitrile group in Y165pCNF is strongly hydrogen bonded such that the

theoretical approach is not applicable. However, in both variants the largest changes of the

nitrile stretching modes occur in the last step of the photoconversion, supporting the view that

the proton-coupled restructuring of the tongue is accompanied by a change of the electric field.

Introduction

Phytochromes are sensory photoreceptors that use the ratio of light intensities as a source of

information to initiate physiological processes.1,2 The light-absorbing unit is a linear methine-

bridged tetrapyrrole which is covalently bound to the protein in the photosensory core module

(PCM) constituted by a PAS-GAF-PHY domain triade.2 The PCM interconverts between the

red absorbing Pr and far-red absorbing Pfr state, in which the chromophore adopts a ZZZssa

and ZZEssa configuration, respectively. Phytochromes are found in plants, fungi, and bacteria.

In eukaryotic and most bacterial phytochromes, Pr is the stable dark state (prototypical

phytochromes), in contrast to bathy bacterial phytochromes, where Pfr is the thermally stable

state. Despite different domain compositions and different types of tetrapyrrole chromophores,

all phytochromes share common features of photoinduced reaction cascades (Figure 1).

Figure 1. Simplified scheme of the photoinduced reactions in phytochromes. Photochemical

and thermal reactions are indicated by red and black arrows, respectively. Further details are

given in the Supporting Information (Fig. S3). The various states of phytochrome with the

chromophore in the ZZZssa (right) and ZZEssa configuration (left) are highlighted by the

yellow and red background color, respectively. The structural formulas also indicate the

localization of important vibrational modes of the chromophore.

The photoinduced conversion between Pr and Pfr starts with double bond photoisomerization

at the methine bridge between rings C and D to yield a distorted, energy-rich ZZEssa (Pr ®

Lumi-R) or ZZZssa (Pfr ® Lumi-F) configuration, presumably with only minor structural

adjustments of the amino acid residues near the chromophore isomerization site.3 The primary

processes are followed by chromophore relaxations and structural rearrangements in the

chromophore binding pocket in the Meta-R and Meta-F states. The decay of the Meta states to

the final photoconversion products is coupled with crucial protein structural changes. This

primarily involves the secondary structure transition of the tongue, a peptide segment in the

PHY domain.4 In prokaryotic phytochromes, this transition represents an important step to

communicate PCM activation or deactivation to the output module, often a histidine kinase.

Particularly detailed insights into the coupling between chromophore and protein structural

changes in the PCM were gained for the bathy phytochrome Agp2 from Agrobacterium fabrum,

based on crystal structures determined for the parent Pfr and the Meta-F states of the wild-type

Agp2-PCM and its variant Agp2-PAiRFP2, and a large body of spectroscopic data (Figure

1).5,6,7,8 The results indicate that E ® Z photoisomerization of the biliverdin (BV; Figure 1)

chromophore leads to reorientations of key amino acid residues in the chromophore binding

pocket, accompanied by a reorganization of the extended hydrogen bond (H-bond) network

around the chromophore. Eventually, a rotation of Tyr165 and Phe192 as well as a removal of

a water molecule on ring D of BV leads to the movement of Gln190 towards Trp440 resulting

in a destabilization of the coil region of the tongue.5 However, a complete conversion of the

tongue from the a-helical to the b-sheet structure requires the proton transfer from the propionic

side chain of BV´s ring C (propC) to His278, which occurs during the decay of Meta-F to Pr.8

On the basis of spectroscopic studies of site-specifically engineered Agp2 variants and Agp2

adducts with BV monomethylester,7,8 we suggested that proton translocation alters the overall

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