| Part ⅠMost quinones are generally classified as toxic compounds that are widely distributed in nature. Cellular accumulation of quinones leads to serious cytotoxic effects in various ways. Quinone dehydrogenase/oxidoreductases protect cells from the cytotoxic effects of quinones. These enzymes catalyze the reduction of quinone to its corresponding hydroquinone, which is readily conjugated to either glucuronic acid or sulfate for excretion. NADPH-dependent quinone oxidoreductases (QORs, EC1.6.5.5) belong to the nonmetallic reductase subfamily of the medium-chain dehydrogenase/reductase superfamily. The ξ-crystallins are NADPH-dependent QORs that were first identified in the lenses of guinea pigs and camels. Initially, it had been reported that ξ-crystallin catalyze one-electron reduction of ortho-quinone. However, recent investigation showed that QOR catalyze two-electron reduction to convert quinone to its corresponding reduced quinone using NADPH as a cofactor. Afterwards, the ξ-crystallin-like QORs have also been identified in other organisms. To date, the structures of human ξ-crystallin, its homologs from Escherichia coli, and Pseudomonas syringae pv. tomato DC3000have been determined. All exist as a homodimer in the asymmetric unit, with each subunit composed of a catalytic domain and a cofactor-binding domain. The ξ-crystallin-like QORs have been biophysically and structurally characterized. However, little is known about their substrate-binding site and catalytic mechanism.The homolog of ξ-crystallin in Saccharomyces cerevisiae is Ztal, and it localizes in both the cytoplasm and the nucleus. Ztal is reported to have similar substrate specificity to human and guinea pig ξ-crystallins. Here, we determined the crystal structures of Ztal, in the apo-form at2.00A and complexed to NADPH at1.59A. Ztal forms a homodimer, with each subunit containing a catalytic and a cofactor-binding domain. Upon NADPH binding to the interdomain cleft, the two domains shift towards each other, producing a better fit for NADPH, and tightening substrate binding. Computational simulation combined with site-directed mutagenesis and enzymatic activity analysis defined a potential quinone-binding site that determines the stringent substrate specificity. Moreover, multiple-sequence alignment and kinetics assays implied that a change in a single residue at the gate of the substrate-binding pocket might cause elevation of enzymatic activity of ξ-crystallin-like QORs from lower to higher organisms. Part ⅡFormation of correct disulfide bonds is important for the structure and function of most proteins. In eukaryotic cells, the intermembrane space (IMS) of mitochondria and endoplasmic reticulum (ER) are the two major places for introduction of disulfide bonds. The IMS has a dedicated disulfide relay system to introduce disulfide bonds into the small cysteine-rich substrate proteins, such as small Tim proteins and copper chaperone Cox17, which are nuclear encoded and cytosolic synthesized. The newly synthesized unfolded substrate proteins would pass through the translocase of the outer membrane and form a mixed disulfide bonded intermediate with Mia40in IMS. Mia40harbors a conserved redox-active motif of-CPC-CX9C-CX9C-, using the CPC site to form an intermolecular disulfide bond with the substrate proteins. Upon the disulfide exchange reaction, a disulfide bond is introduced into the substrate protein, accompanying with the release of the reduced Mia40, which is in turn regenerated/reoxidized to a functional state by sulfhydryl oxidase Ervl. Thereafter, the reduced Ervl forwards the electron to either cytochrome c or molecular oxygen. Together, Mia40and Ervl are two essential components of the disulfide relay system that is of crucial importance for mitochondrial biogenesis.The flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidase Ervl (EC1.8.3.2) is first recognized as a protein essential for the respiration and vegetative growth of the yeast. Afterwards, a number of Ervl homologs were characterized in plants, mammals and double-stranded DNA viruses. The mammalian homologs were also called ALRs, for augmenters of liver regeneration. All Erv1/ALR family members share a conserved core domain harboring a motif of CXXC (the core redox-center), which is juxtaposed with FAD and involved in redox reaction. To date, the structures of the core domain of human ALR, Arabidopsis thaliana Ervl, Rattus norvegicus ALR, and Saccharomyces cerevisiae Erv2have been determined. All exist as a homodimer, with each subunit composed of a four-helical bundle that accommodates the isoalloxazine ring of FAD and an additional single-turn helix. In addition to the conserved core domain, Ervl/ALR proteins, except for the viral homologs, possess another cysteine pair at the N-terminal region in fungi and animals or at the C-terminal segment in plants. Genetic studies demonstrated that the N-terminal CXXC motif of yeast Ervl was required for the in vivo functions. In fact, the N-terminal region of yeast Ervl is necessary and sufficient for the interaction with Mia40, and moreover the N-terminal cysteine pair is required for the formation of a mixed-disulfide intermediate with Mia40. Thanks to its role in forwarding electrons from Mia40to the C-terminal core domain (CTD), the CXXC motif of the N-terminal region is termed the "shuttle" redox-center.To gain insights into the structural basis of this electron transfer chain, we determined the structures of the CTD and Ervlm (a C30S/C133S double mutant of Erv1) at2.0A and3.0A resolution, respectively. Similar to the previous structures, the CTD exists as a homodimer, each subunit of which consists of a conserved four-helical helix bundle and an additional single-turn helix. The structure of Erv1m enabled us to identify, for the first time, the three-dimensional structure of N-terminal shuttle region, which is composed of one N-terminal shuttle domain (NTD) and one flexible loop. This structure also captures the intermediate state of electron transfer from one NTD to the CTD of another subunit. Comparative structural analysis revealed significant conformational changes of the four-helix bundle of CTD, which form a "receiver" to harbor the electron holder NTD. Moreover, computational simulation combined with multiple-sequence alignment demonstrated the amphipathic helix downstream of shuttle redox-center is critical for the recognition of upstream electron donor Mia40. Taken together, these findings provide structural insights into the electron transfer from Mia40, via the NTD of one subunit, to the CTD of another subunit of Ervl. |
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