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Lothrop, Adam P.

Biochemistry

2012

PhD

High Mr thioredoxin reductases (TR) are members ofthe pyridine nucleotide disulfide oxidoreductase family of enzymes. Functionally homodimeric, TR transfers reducing equivalents from NADPH to a bound FAD molecule, which then reduces a redox active disulfide/dithiol pair near the N-terminus of the enzyme. A C-terminal, tail-like extension from the opposite monomer containing a redox active vicinal disulfide is reduced by the N-terminal redox center that can subsequently pass electrons to the TR substrate, thioredoxin. Disulfide bonds that form from adjacent (vicinal) cysteine (Cys) residues are an unusual occurrence in proteins, resulting in an 8-membered ring structure. Some TRs (such as those ofmammals) utilize the rare, selenium containing amino acid selenocysteine (Sec, U) as part of their C-terminal redox center, while TRs of many other organisms use the standard Cys residue in the enzyme. Both Cys-and Sec-TRs are known to reduce thioredoxin 'with comparable catalytic efficiency, which raises the question of why some organisms preserve a complicated, energetically costly insertion system for Sec when Cys can perform the same function in the enzyme.
The aims of this project were to determine the function of Se in the mechanism of Sec-TRs (the mechano-enzymatic function) by examining how Cys-TRs compensate for the absence of this rare amino acid. To that end, we used mouse mitochondrial TR (mTR3, a Sec-TR) and cytosolic TR from Drosophi/a melanogaster (DmTR, a Cys-TR) as representatives of each TR type and probed the mechanisms of these enzymes. A major finding with respect to the mechano-enzymatic function of Se in mTR3 was that the N-terminal redox center of this enzyme can reduce a variety of different substrates with strong electrophilic character. As this includes many Se-containing substrates, we concluded that Se must be functioning as a superior acceptor of electrons during the transfer of electrons between N-and C-terminal redox centers (which we believe is rate limiting in the TR mechanism) due to its intrinsic electrophilicity.
Building on previous experiments that showed DmTR required a specific geometry imparted by the 8-membered ring of its oxidized C-terminal redox center in order for electron transfer between redox centers to occur, we increased the size of the ring by insertion of alanine residues in between the vicinal Cys or mutation of Cys to homocysteine (hCys). Replacement of the penultimate Cys residue with hCys did not reduce activity as much as expected. Further investigation indicated that we had uncovered an alternative mechanism that allowed DmTR with hCys as its penultimate residue to reduce Trx without undergoing ring formation. Based upon our DmTR data and the new hypothesis of Se as a superior electrophile in facilitating the exchange step between N-and C-terminal redox centers in Sec-TRs we propose an "electrophilic activation" mechanism whereby a Cys-TR can polarize the disulfide bond of its oxidized C-terminal redox center to make the S atom ofthe Cys residue more electron deficient (similar to Se in Sec) to enhance the rate ofthe exchange step in the TR mechanism. The ability of theC-terminal redox centers of both Sec-andCys-TRs to accept electronsfrom the N-terminal redox center is crucial to the mechanism of these enzymes.