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

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. 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-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.

Pushing the limits of flash photolysis to unravel the secrets of biological electron and proton transfer

“…We are happy to announce that today, this virtual volume unites 18 contributions by scientists from 17 countries (Canada, Czech republic, Finland, France, Germany, Hungary, India, Italy, Japan, Lebanon, Poland, Russia, Spain, Switzerland, Taiwan, UK, US) touching upon the wide variety of the subjects dealt with by Klaus at various times [25,[127][128][129][130][131][132][133][134][135][136][137][138][139][140][141][142][143].…”

“…Gindt et al studied the oxidation of native semiquinone FADH° in VcCRY-1, a member of the cryptochrome DASH family, whose flavoproteins may use blue light both for repairing CPDs and for signal transduction [136]. The authors found both pH and substrate-binding-dependent oxidation kinetics.…”

The Impact of the Functional Layer Composition of Glucose Test-Strips on the Stability of Electrochemical Response

“…In contrast, the highest sensitivity to glucose test-strips with the dried sensing layer was obtained with the gel prepared in citrate buffer (Figure 2b,c). The observed difference in enzyme activity could be explained in terms of different interaction rates between oxygen and reduced cofactor FAD/FADH2 of GOx in solution (see Reactions (1) and ( 2) and the same reactions in the presence of RedOx mediator in the gels [37]):…”

“…At the high glucose concentration, the use of PPBI buffer with pH 7.4 and AcB with pH 5.5 led to a significant current decrease, especially for AS containing gels. The reason The observed difference in enzyme activity could be explained in terms of different interaction rates between oxygen and reduced cofactor FAD/FADH 2 of GOx in solution (see Reactions (1) and ( 2) and the same reactions in the presence of RedOx mediator in the gels [37]):…”