Biliverdin IXβ reductase S¹¹¹L characterization. (A) BLVRB expression in gel-filtered platelets (10 μg/lane) from representative cohorts that are wild type (^−/−) or heterozygous (^+/−) for mutant BLVRB^462C→T. (B) Schematic globular structure displays BLVRB^S111L mutation (green) within the single BV/NAD(P)H binding fold, with higher-resolution ribbon structures (insets) modeled to predict mutant Leu¹¹¹ or native Ser¹¹¹ on BV or NADPH interactions; note that Ser¹¹¹ is uniquely positioned for recognition and/or proton transfer within the Rossmann binding fold, with predicted proximity interference by the hydrophobic Leu¹¹¹ aliphatic isobutyl side chain. Models were generated using PYMOL software,²⁸  based on NADP/mesobiliverdin IVα ternary (Protein Data Bank ID code 1HE3) and NADP/flavin mononucleotide ternary (Protein Data Bank ID code 1HE4) complexes. (C) Immunoblot of purified recombinant enzymes after thrombin cleavage and glutathione affinity depletion of the GST (glutathione-S-transferase) carrier (50 ng/lane). (D-E) Recombinant BLVRB^WT and BLVRB^S111L were used for flavin reductase (100 μM flavin mononucleotide) or BVR (20 μM BV dimethyl esters) specific activity determinations¹⁵  (N = 6, expressed as mean ± SEM); **** P < .00001. (F) Solubilized lysates from Lv-infected HEK293 cells were used for BVR activity in the presence of 20 μM BV dimethyl esters (N = 3); *P < .05; immunoblot of Lv-infected HEK293 cells (20 μg/lane) is shown. (G) BVR activity assays using recombinant BLVRB^WT or commercially available BLVRA.