Biological sulfur in the blood cells of Ascidia ceratodes: XAS spectroscopy and a cellular-enzymatic hypothesis for vanadium reduction in the ascidians

Two samples of living blood cells and of cleared blood plasma from the Phlebobranch tunicate Ascidia ceratodes from Bodega Bay, California, and one of fresh Henze solution from A. ceratodes of Monterey Bay, California, have been examined using sulfur K-edge x-ray absorption spectroscopy (XAS). Biological sulfur included sulfate esters, sulfate and bisulfate ions, benzothiazole, thianthrene, epi-sulfide, thiol and disulfide. Glutathione dominated reduced sulfur, from which an average intracellular Voltage of −0.21 V was calculated. Sulfate-bisulfate ratios yielded blood cell pH values of 2.0 and 2.8. Total blood cell [sulfur] was 373±9 mM or 296±73 mM from BaSO4 gravimetry. Two plasma samples (pH 6.9 or 7.0; [S] = 33±6 mM or 26±4 mM) were dominated by sulfate and disulfide. Fresh Henze solution evidenced a sulfur inventory similar to blood cells, with calculated pH = 2.7. A V(III)-sulfonate fraction varied systematically with intracellular pH across six independent blood cell samples, implying a vanadium mobilization pathway. Bodega Bay and Monterey Bay A. ceratodes appear to maintain alternative suites of low-valent sulfur. The significance of the vanabins to vanadium metabolism is critically examined in terms of known protein – V(IV) biochemistry. Finally, a detailed hypothesis for the reduction of [VO4]3− to V(III) in ascidians is introduced. A vanadium oxido-reductase is proposed to span the signet ring membrane and to release V(III) into the inner acidic vacuole. The V(V) to V(III) reduction is predicted require an inner-sphere mechanism, a thiol reductant, 7-coordinate V(III), a biologically accessible Voltage, and proton-facilitated release of V(III). Sulfur K-edge XAS spectra of blood cells from Ascidia ceratodes were fit with model XAS spectra. Low valent sulfur was dominated by the glutathiol/glutathione couple. Thianthrene, benzothiophene, sulfate, bisulfate, and sulfate esters were all detected. Derived intracellular Voltage and pH were −0.21 V, and 2.0 and 2.8, respectively. [Display omitted] •Glutathiol/glutathione governs blood cell Voltage in Bodega Bay Ascidia ceratodes.•Average blood cell Voltage was −0.21 V and signet ring vacuolar pH was 2.0 or 2.8.•The biological reduction of V(V) to V(III) is an inner-sphere electron transfer.•VO2+-protein interactions are insufficient to indicate biological significance.•The enzymatic active site, mechanism, and location of V(V) reduction are proposed.