Role of cysteine residues in the V(V)-reductase activity of Vanabin2

Abstract

Ascidians (tunicates or sea squirts) accumulate extremely high levels of vanadium as the reduced form V(III) in extremely acidic vacuoles in their blood cells. Several key proteins related to vanadium accumulation have been isolated from vanadium-rich ascidians and their physiological functions characterized. Of these, vanabins are small, cysteine-rich proteins that have been identified only in vanadium-rich ascidians. Our previous study revealed that Vanabin2 can act as a V(V)-reductase. The current study examines the role of cysteine and several other amino acid residues of Vanabin2 in V(V)-reduction. When all eighteen cysteine residues of Vanabin2 were substituted with serine residues, the V(V)-reductase activity was lost. Substitutions of three, structurally clustered cysteines in three different regions resulted in a moderate decrease in reductase activity, suggesting that more than a single cysteine pair is responsible for the V(V)-reductase activity of Vanabin2. Mutations in the V(IV)-binding domains caused either an increase or decrease in activity but no mutation caused the complete 2 loss of activity. These results suggest that some pairs, but more than a single pair, of cysteine residues are necessary for the V(V)-reductase activity of Vanabin2.

Vanadium accumulation in ascidians: A system overview

“…Vanabin2 [81]. These results suggest that electrons may be transferred from the donor NADPH to the acceptor V V ions via thiol-disulfide exchange reactions in Vanabin2 (Fig.…”

Participation of thioredoxin in the V(V)-reduction reaction by Vanabin2

“…and V(V) [17]. Here, we determined that the cascade including AsTrx1 is also dependent on cysteine residues in Vanabin2.…”

“…Recombinant wild-type (WT) and mutant Vanabin2 proteins were prepared in accordance with procedures published previously [7,17]. Preparation of the AsTrx1 protein was essentially identical to the published procedures.…”

A Role for Vanadium in Ascidians and in Marine Algae

“…The intermittently formed VO 2+ binds to the lysine NH 2 residues of the vanabins. Vanabins are lysine-rich polypeptides of about 10 thousand kDa, attaining a bow-shaped conformation, with four α-helices connected by nine disulfide bonds [6][7][8]. The amount of vanadium accumulated by ascidians strongly differs.…”

“…Vanadate (V) enters the cell via phosphate/vanadate channels, supported by mutualistic bacteria. Vanadium binding sites are provided by lysine-rich polypeptides [6,9]. The blood cells storing V 3+ are termed vanadocytes.…”