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Tolentino Collado, Jinnette, Iuliano, James N., Pirisi, Katalin, Jewlikar, Samruddhi, Adamczyk, Katrin, Greetham, Gregory M., Towrie, Michael, Tame, Jeremy R. H., Meech, Stephen R. 
ORCID: https://orcid.org/0000-0001-5561-2782, Tonge, Peter J. and Lukacs, Andras
(2022)
Unraveling the photoactivation mechanism of a light activated adenylyl cyclase using ultrafast spectroscopy coupled with unnatural amino acid mutagenesis.
ACS Chemical Biology, 17 (9).
2643–2654.
ISSN 1554-8929
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Abstract
The hydrogen bonding network that surrounds the flavin in Blue Light Utilizing FAD (BLUF) photoreceptors plays a crucial role in sensing and communicating the changes in the electronic structure of the flavin to the protein matrix upon light absorption. The network contains a highly conserved tyrosine that is essential for photoactivation. Using time-resolved infrared spectroscopy (TRIR) and unnatural amino acid (UAA) incorporation, we investigated the photoactivation mechanism and the role of the conserved tyrosine (Y6) in the forward reaction of the photoactivated adenylyl cyclase (AC) from Oscillatoria Acuminata (OaPAC). Our work elucidates the direct connection between the photoactivation process in the BLUF domain and the structural and functional implications on the partner protein for the first time. The TRIR results demonstrate formation of FADH● as an intermediate species on the photoactivation pathway which decays to form the signaling state. Using fluorotyrosine analogs to modulate the physical properties of Y6, the TRIR data reveal that a change in the pKa and/or reduction potential of Y6 has a profound effect on the forward reaction, consistent with a mechanism involving proton transfer or proton-coupled electron transfer from Y6 to the electronically excited FAD. Decreasing the pKa from 9.9 to <7.2 and/or increasing the reduction potential by 200 mV of Y6 prevents proton transfer to the flavin and halts the photocycle at FAD● ̶. The lack of protonation of the anionic flavin radical can be directly linked to photoactivation of the AC domain. While the 3F-Y6 and 2,3-F2Y6 variants undergo the complete photocycle and catalyze the conversion of ATP to cAMP, enzyme activity is abolished in the 3,5-F2Y6 and 2,3,5-F3Y6 variants where the photocycle is halted at FAD● ̶. Our results thus show that proton transfer plays an essential role in initiating the structural reorganization of the AC domain that results in adenylyl cyclase activity.
| Item Type: | Article |
|---|---|
| Additional Information: | Acknowledgments: J.T.C. was supported by the IMSD-MERGE and NY-CAPs IRACDA (K12-GM102778) Programs at Stony Brook University. A.L. acknowledges funding from the Hungarian National Research and Innovation Office (K-137557) and was supported by PTE ÁOK-KA-2021. This study was supported by the National Science Foundation (NSF) (MCB-1817837 to PJT) and the EPSRC (EP/N033647/1 to S.R.M.). J.N.I was supported by a National Institutes of Health Chemistry-Biology Interface Training Grant (T32GM092714). The authors are grateful to STFC for access to the ULTRA laser facility. |
| Uncontrolled Keywords: | molecular medicine,biochemistry,2*,good paper but similar to earlier work. ,/dk/atira/pure/subjectarea/asjc/1300/1313 |
| Faculty \ School: | Faculty of Science > School of Chemistry (former - to 2024) |
| UEA Research Groups: | Faculty of Science > Research Groups > Chemistry of Light and Energy Faculty of Science > Research Groups > Centre for Photonics and Quantum Science |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 06 Sep 2022 11:48 |
| Last Modified: | 25 Sep 2024 16:40 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/87725 |
| DOI: | 10.1021/acschembio.2c00575 |
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