a, Purified Synechococcus protein, containing the VKOR and Trx-like domains, was incubated with reduced, denatured RNAse A and vitamin K1 (Vit K1) (lanes 9, 10). The reaction was quenched with AMS, which modifies free sulfhydryl groups. Samples were analyzed by SDS-PAGE and Coomassie staining. Controls were performed without Vit K1 (in ethanol, EtOH) or VKOR, as indicated. RNAse A was also oxidized with glutathione (GSH/GSSG) (lanes 3,4). b, As in a, but with VKOR serially diluted. c, As in a, but with wild type (WT) or mutants in cysteines in the predicted electron transfer pathway (Fig. 4). d, As in c, except reduction of Vit K1 was followed in a fluorometer, using reduced RNAse or dithiothreitol (DTT) as reductant. Error bars represent standard error of mean instrumental fluorescence variation.