Site-specific insertion of selenium into the redox-active disulfide of the flavoprotein augmenter of liver regeneration

Abstract

Augmenter of liver regeneration (sfALR) is a small disulfide-bridged homodimeric flavoprotein with sulfhydryl oxidase activity. Here, we investigate the catalytic and spectroscopic consequences of selectively replacing C145 by a selenocysteine to complement earlier studies in which random substitution of ~90% of the 6 cysteine residues per sfALR monomer was achieved growing Escherichia coli on selenite. A selenocysteine insertion sequence (SECIS) element was installed within the gene for human sfALR. SecALR2 showed a spectrum comparable to that of wild-type sfALR. The catalytic efficiency of SecALR2 towards dithiothreitol was 6.8-fold lower than a corresponding construct in which position 145 was returned to a cysteine residue while retaining the additional mutations introduced with the SECIS element. This all-cysteine control enzyme formed a mixed disulfide between C142 and β-mercaptoethanol releasing C145 to form a thiolate-flavin charge transfer absorbance band at ~530 nm. In contrast, SecALR2 showed a prominent long-wavelength absorbance at 585 nm consistent with the expectation that a selenolate would be a better charge-transfer donor to the isoalloxazine ring. These data show the robustness of the ALR protein fold towards the multiple mutations required to insert the SECIS element and provide the first example of a selenolate to flavin charge-transfer complex.

Spectroscopic evidence for direct flavin-flavin contact in a bifurcating electron transfer flavoprotein

“…Meanwhile the Bf-flavin is not mobile relative to residue 37 and is 12 Å away from the nearest aromatic side chain based on our model and available structures (14). Cys is competent to form a CT complex with OX Bf-flavin, especially upon ionization (76,77). However C174.L was proven not to be required for 726 nm species formation by our C174A.L variant.…”

Turning gray selenium into a nanoaccelerator of tissue regeneration by PEG modification

“…Epidemiological studies have suggested the correlation of Se deficiency and poor prognosis of the spinal cord [ 46 ] and liver regeneration [ 47 ]. However, there is still no direct evidence that selenium benefits tissue regeneration.…”

Synthesis, structures and electronic properties of Co(III) complexes with 2-quinolinecarboxaldehyde thio- and selenosemicarbazone: A combined experimental and theoretical study

“…A better understanding of the nature and consequences of this substitution were the main goals of many recent biochemical and theoretical studies. 22,23 Herein, the syntheses and crystal structures of Co(III) complexes with thiosemicarbazone and selenosemicarbazone ligands are reported. To obtain detailed characterization of these compounds, which could provide a better insight into the structure-activity relationship, cyclic voltammetry, NMR and UV/Vis spectroscopic analyses and the measurement of antioxidant capacity are presented together with a theoretical study.…”