Investigation of the pH-dependence of the oxidation of FAD in VcCRY-1, a member of the cryptochrome-DASH family

Yvonne M. Gindt, Gabrielle Connolly, Amy L. Vonder Haar, Miryam Kikhwa, Johannes P.M. Schelvis

Research output: Contribution to journalArticlepeer-review

Abstract

Vibrio cholerae cryptochrome-1 (VcCRY-1) is a member of the cryptochrome DASH family. The flavoprotein 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 pKa ≈ 7.0. Stopped-flow 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 flow, but there was only a small effect 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-effect 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-effect.

Original languageEnglish
Pages (from-to)831-841
Number of pages11
JournalPhotochemical and Photobiological Sciences
Volume20
Issue number6
DOIs
StatePublished - Jun 2021

Keywords

  • Absorption spectroscopy
  • Cryptochrome-DASH
  • DNA repair
  • FAD oxidation
  • Kinetics
  • Raman spectroscopy

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