In 2009, Comi and coworkers (Di Fonzo et al., PubMed) identified the point mutation that caused three children of consanguineous parents to develop cataract, progressive muscle weakness, hearing loss and developmental delay. The mutant protein was a mitochondrial sulfhydryl oxidase: augmenter of liver regeneration (here abbreviated ALR). The long form of ALR is resident in the mitochondrial intermembrane space (IMS) and participates in the disulfide-generating pathway shown below. ALR (depicted in green) receives reducing equivalents from Mia40 and passes them to either cytochrome c or, directly, to molecular oxygen.
Di Fonzo et al. identified the mutation: an arginine at position 194 is replaced by a histidine. They combined cell-based studies with a recapitulation of the R194H mutation in the yeast counterpart of ALR – a protein called Erv1p. Overall, they concluded that the mutation affected the import and/or stability of ALR within the IMS.
Daithankar et al. (PubMed) have recently extended this work with a determination of the crystal structure of the catalytic core domain of human ALR (PDB) and a characterization of the R194H mutation on the in vitro activity and stability of the long (IMS form) and short (cytokine-like) forms of the enzyme. The mutation does not affect enzymatic activity of human ALR, but significantly decreases the thermal stability, increases the conformational flexibility of regions of the mutant protein and weakens the binding of FAD. Indeed, the side chain of R194 forms multiple H-bonds in the vicinity of the ribose moiety of FAD in the wild-type protein – a web of interactions that involve the subunit interface that cannot be fulfilled by the histidine mutant.
Human ALR (like the rat protein – PDB) is a covalent homo-dimeric flavoprotein (the structures for both mammalian proteins represent the short form of the protein). Head to tail disulfides link the grey and green subunits close to their N- and C-terminii. The arginine 194 residue is shown in the inset. The human structure was determined in collaboration with Ming Dong and Brian Bahnson.
These structural and biochemical studies on human ALR confirm and extend the work of Comi and colleagues on this first human sulfhydryl oxidase mutant to be described. While the current work has focussed on the enzymatic activity and stability of the protein in the mitochondrial IMS, the possible impact of the R194H mutant on the role of ALR as a cytokine remain to be investigated.