Vol.:(0123456789) 1 3 Photochemical & Photobiological Sciences (2021) 20:831–841 https://doi.org/10.1007/s43630-021-00063-5 ORIGINAL PAPERS Investigation of the pH‑dependence of the oxidation of FAD in VcCRY‑1, a member of the cryptochrome‑DASH family Yvonne M. Gindt 1  · Gabrielle Connolly 1  · Amy L. Vonder Haar 1  · Miryam Kikhwa 1  · Johannes P. M. Schelvis 1 Received: 1 March 2021 / Accepted: 31 May 2021 / Published online: 6 June 2021 © The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology 2021 Abstract Vibrio cholerae cryptochrome-1 (VcCRY-1) is a member of the cryptochrome DASH family. The favoprotein appears to use blue light both for repair of cyclobutane pyrimidine dimers (CPDs) on DNA and signal transduction. Earlier, we found that it was almost impossible to oxidize the FADH· state upon binding to a CPD, and, in the absence of substrate, the rate of FADH· oxidation was much larger at high pH (Gindt et al. in Biochemistry 54:2802–2805, 2015). Here, we present the pH-dependence of the oxidation of FADH· by ferricyanide, which revealed a switch between slow and fast oxidation with a pK a ≈ 7.0. Stopped-fow mixing was used to measure the oxidation of FADH − to FADH· at pH 6.7 and 7.5. Substrate binding was required to slow down this oxidation such that it could be measured with stopped fow, but there was only a small efect of pH. In addition, resonance Raman measurements of FADH· in VcCRY-1 at pH 6.5 and 7.5 were performed to probe for structural changes near the FAD cofactor related to the observed changes in rate of FADH· oxidation. Only substrate binding seemed to induce a change near the FAD cofactor that may relate to the change in oxidation kinetics. The pH-efect on the FADH· oxidation rate, which is rate-limited by the proton acceptor, does not seem to be due to a protein structural change near the FAD cofactor. Instead, a conserved glutamate in CRY-DASH may control the deprotonation of FADH· and give rise to the pH-efect. Keywords Cryptochrome-DASH · DNA repair · FAD oxidation · Kinetics · Absorption spectroscopy · Raman spectroscopy * Johannes P. M. Schelvis schelvisj@montclair.edu 1 Department of Chemistry and Biochemistry, Montclair State University, Montclair, NJ 07043, USA Pushing the limits of fash photolysis to unravel the secrets of biological electron and proton transfer - a topical issue in honour of Klaus Brettel. 1 Introduction The cryptochrome-DASH (CRY-DASH) subfamily is a member of the cryptochrome/photolyase family (CPF) of favoproteins [1–5]. In addition to CRY-DASH proteins, the CPF is further comprised of cyclobutane pyrimidine dimer (CPD) and (6–4)–photolyases (PL) that repair CPDs and (6–4)–pyrimidine pyrimidone photoproducts on DNA, respectively, along with the plant and animal cryptochromes (CRY) that are involved in signaling in plant growth and regulation of the circadian clock [2–7]. All members of the CPF bind the required active site favin adenine dinucleotide (FAD) cofactor in their N-terminal domain, and, in addition, many members of the CPF family bind a second chromo- phore that seems to act as a light-gathering antenna [3]. DNA binding and repair occur at the N-terminal domain in PLs and CRY-DASH. Although CRYs have a photolyase homology domain in their N-terminal domain, they do not repair DNA. The CRYs do possess a C-terminal domain that varies in length and sequence between CRYs and is impor- tant for their signaling function. CRY-DASH has retained a non-canonical DNA-repair function but also has a signaling function, and is believed to be an evolutionary intermediate between PL and CRY [8]. All members of CPF use light absorption by the required FAD cofactor for their function, though CRYs in higher animals can be involved in entrainment of the circadian rhythm in a light-independent way [2–7]. In PL, the active state is the fully reduced FADH − , and the enzyme cycles through the neutral radical FADH· state during its photo- catalytic repair function [3]. PL in the FADH· state can be photoactivated (i.e. photoreduced) by light to form the active FADH – state, though the fully oxidized FAD (FAD ox ) state