| Abstract
| - Methemoglobinemia, the first hereditary disease to be identified that involved an enzymedeficiency, has been ascribed to mutations in the enzyme cytochrome b5 reductase. A variety of defectsin either the erythrocytic or microsomal forms of the enzyme have been identified that give rise to thetype I or type II variant of the disease, respectively. The positions of the methemoglobinemia-causingmutations are scattered throughout the protein sequence, but the majority of the nontruncated mutantsthat produce type II symptoms occur close to the flavin adenine dinucleotide (FAD) cofactor binding site.While X-ray structures have been determined for the soluble, flavin-containing diaphorase domains ofthe rat and pig enzymes, no X-ray or NMR structure has been described for the human enzyme or any ofthe methemoglobinemia variants. S127P, a mutant that causes type II methemoglobinemia, was the firstto be positively identified and have its spectroscopic and kinetic properties characterized that revealedaltered nicotinamide adenine dinucleotide hydride (NADH) substrate binding behavior. To understandthese changes at a structural level, we have determined the structure of the S127P mutant of rat cytochromeb5 reductase to 1.8 Å resolution, providing the first structural snapshot of a cytochrome b5 reductasemutant that causes methemoglobinemia. The high-resolution structure revealed that the adenosinediphosphate (ADP) moiety of the FAD prosthetic group is displaced into the corresponding ADP bindingsite of the physiological substrate, NADH, thus acting as a substrate inhibitor which is consistent withboth the spectroscopic and kinetic data.
|
