Augmenter of Liver Regeneration: Substrate Specificity of a Flavin-Dependent Oxidoreductase from the Mitochondrial Intermembrane Space

Augmenter of liver regeneration (ALR) is both a growth factor and a sulfhydryl oxidase that binds FAD in an unusual helix-rich domain containing a redox-active CxxC disulfide proximal to the flavin ring. In addition to the cytokine form of ALR (sfALR) that circulates in serum, a longer form, lfALR, is believed to participate in oxidative trapping of reduced proteins entering the mitochondrial intermembrane space (IMS). This longer form has an 80-residue N-terminal extension containing an additional, distal, CxxC motif. This work presents the first enzymological characterization of human lfALR. The N-terminal region conveys no catalytic advantage toward the oxidation of the model substrate dithiothreitol (DTT). In addition, a C71A or C74A mutation of the distal disulfide does not increase the turnover number toward DTT. Unlike Erv1p, the yeast homologue of lfALR, static spectrophotometric experiments with the human oxidase provide no evidence of communication between distal and proximal disulfides. An N-terminal His-tagged version of human Mia40, a resident oxidoreductase of the IMS and a putative physiological reductant of lfALR, was subcloned and expressed in Escherichia coli BL21 DE3 cells. Mia40, as isolated, shows a visible spectrum characteristic of an Fe-S center and contains 0.56 +/- 0.02 atom of iron per subunit. Treatment of Mia40 with guanidine hydrochloride and triscarboxyethylphosphine hydrochloride during purification removed this chromophore. The resulting protein, with a reduced CxC motif, was a good substrate of lfALR. However, neither sfALR nor lfALR mutants lacking the distal disulfide could oxidize reduced Mia40 efficiently. Thus, catalysis involves a flow of reducing equivalents from the reduced CxC motif of Mia40 to distal and then proximal CxxC motifs of lfALR to the flavin ring and, finally, to cytochrome c or molecular oxygen.