Electrochemical evidence that pyranopterin redox chemistry controls the catalysis of YedY, a mononuclear Mo enzyme

A long-standing contradiction in the field of mononuclear Mo enzyme research is that small-molecule chemistry on active-site mimic compounds predicts ligand participation in the electron transfer reactions, but biochemical measurements only suggest metal-centered catalytic electron transfer. With the simultaneous measurement of substrate turnover and reversible electron transfer that is provided by Fourier-transformed alternating-current voltammetry, we show that Escherichia coli YedY is a mononuclear Mo enzyme that reconciles this conflict. In YedY, addition of three protons and three electrons to the well-characterized "as-isolated" Mo(V) oxidation state is needed to initiate the catalytic reduction of either dimethyl sulfoxide or trimethylamine N-oxide. Based on comparison with earlier studies and our UV-vis redox titration data, we assign the reversible one-proton and one-electron reduction process centered around +174 mV vs. standard hydrogen electrode at pH 7 to a Mo(V)-to-Mo(IV) conversion but ascribe the two-proton and two-electron transition occurring at negative potential to the organic pyranopterin ligand system. We predict that a dihydro-to-tetrahydro transition is needed to generate the catalytically active state of the enzyme. This is a previously unidentified mechanism, suggested by the structural simplicity of YedY, a protein in which Mo is the only metal site

Simultaneous recording of multiple cellular signaling events by frequency- and spectrally-tuned multiplexing of fluorescent probes

Fluorescent probes that change their spectral properties upon binding to small biomolecules, ions, or changes in the membrane potential (V(m)) are invaluable tools to study cellular signaling pathways. Here, we introduce a novel technique for simultaneous recording of multiple probes at millisecond time resolution: frequency- and spectrally-tuned multiplexing (FAST(M)). Different from present multiplexing approaches, FAST(M) uses phase-sensitive signal detection, which renders various combinations of common probes for V(m) and ions accessible for multiplexing. Using kinetic stopped-flow fluorimetry, we show that FAST(M) allows simultaneous recording of rapid changes in Ca(2+), pH, Na(+), and V(m) with high sensitivity and minimal crosstalk. FAST(M) is also suited for multiplexing using single-cell microscopy and genetically encoded FRET biosensors. Moreover, FAST(M) is compatible with optochemical tools to study signaling using light. Finally, we show that the exceptional time resolution of FAST(M) also allows resolving rapid chemical reactions. Altogether, FAST(M) opens new opportunities for interrogating cellular signaling

A novel cross-species inhibitor to study the function of CatSper Ca 2 + channels in sperm

Background and PurposeSperm from many species share the sperm‐specific Ca2+ channel CatSper that controls the intracellular Ca2+ concentration and, thereby, the swimming behaviour. A growing body of evidence suggests that the mechanisms controlling the activity of CatSper and its role during fertilization differ among species. A lack of suitable pharmacological tools has hampered the elucidation of the function of CatSper. Known inhibitors of CatSper exhibit considerable side effects and also inhibit Slo3, the principal K+ channel of mammalian sperm. The compound RU1968 was reported to suppress Ca2+ signaling in human sperm by an unknown mechanism. Here, we examined the action of RU1968 on CatSper in sperm from humans, mice, and sea urchins.Experimental ApproachWe resynthesized RU1968 and studied its action on sperm from humans, mice, and the sea urchin Arbacia punctulata by Ca2+ fluorimetry, single‐cell Ca2+ imaging, electrophysiology, opto‐chemistry, and motility analysis.Key ResultsRU1968 inhibited CatSper in sperm from invertebrates and mammals. The compound lacked toxic side effects in human sperm, did not affect mouse Slo3, and inhibited human Slo3 with about 15‐fold lower potency than CatSper. Moreover, in human sperm, RU1968 mimicked CatSper dysfunction and suppressed motility responses evoked by progesterone, an oviductal steroid known to activate CatSper. Finally, RU1968 abolished CatSper‐mediated chemotactic navigation in sea urchin sperm.Conclusion and ImplicationsWe propose RU1968 as a novel tool to elucidate the function of CatSper channels in sperm across species