Effect of exchange of the cysteine molybdenum ligand with selenocysteine on the structure and function of the active site in human sulfite oxidase
(2013) In Biochemistry 52(46). p.8295-8303- Abstract
Sulfite oxidase (SO) is an essential molybdoenzyme for humans, catalyzing the final step in the degradation of sulfur-containing amino acids and lipids, which is the oxidation of sulfite to sulfate. The catalytic site of SO consists of a molybdenum ion bound to the dithiolene sulfurs of one molybdopterin (MPT) molecule, carrying two oxygen ligands, and is further coordinated by the thiol sulfur of a conserved cysteine residue. We have exchanged four non-active site cysteines in the molybdenum cofactor (Moco) binding domain of human SO (SOMD) with serine using site-directed mutagenesis. This facilitated the specific replacement of the active site Cys207 with selenocysteine during protein expression in Escherichia coli. The sulfite... (More)
Sulfite oxidase (SO) is an essential molybdoenzyme for humans, catalyzing the final step in the degradation of sulfur-containing amino acids and lipids, which is the oxidation of sulfite to sulfate. The catalytic site of SO consists of a molybdenum ion bound to the dithiolene sulfurs of one molybdopterin (MPT) molecule, carrying two oxygen ligands, and is further coordinated by the thiol sulfur of a conserved cysteine residue. We have exchanged four non-active site cysteines in the molybdenum cofactor (Moco) binding domain of human SO (SOMD) with serine using site-directed mutagenesis. This facilitated the specific replacement of the active site Cys207 with selenocysteine during protein expression in Escherichia coli. The sulfite oxidizing activity (k cat/KM) of SeSOMD4Ser was increased at least 1.5-fold, and the pH optimum was shifted to a more acidic value compared to those of SOMD4Ser and SOMD4Cys(wt). X-ray absorption spectroscopy revealed a MoVI-Se bond length of 2.51 Å, likely caused by the specific binding of Sec207 to the molybdenum, and otherwise rather similar square-pyramidal S/Se(Cys)O2MoVIS 2(MPT) site structures in the three constructs. The low-pH form of the Mo(V) electron paramagnetic resonance (EPR) signal of SeSOMD4Ser was altered compared to those of SOMD4Ser and SOMD 4Cys(wt), with g1 in particular shifted to a lower magnetic field, due to the Se ligation at the molybdenum. In contrast, the Mo(V) EPR signal of the high-pH form was unchanged. The substantially stronger effect of substituting selenocysteine for cysteine at low pH as compared to high pH is most likely due to the decreased covalency of the Mo-Se bond.
(Less)
- author
- Reschke, Stefan ; Niks, Dimitri ; Wilson, Heather ; Sigfridsson, Kajsa G.V. LU ; Haumann, Michael ; Rajagopalan, K. V. ; Hille, Russ and Leimkühler, Silke
- publishing date
- 2013-11-19
- type
- Contribution to journal
- publication status
- published
- in
- Biochemistry
- volume
- 52
- issue
- 46
- pages
- 9 pages
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- scopus:84888209794
- pmid:24147957
- ISSN
- 0006-2960
- DOI
- 10.1021/bi4008512
- language
- English
- LU publication?
- no
- id
- c1514c17-a80f-4cad-b2a5-b297be04d53d
- date added to LUP
- 2020-01-15 10:15:11
- date last changed
- 2024-05-01 04:09:49
@article{c1514c17-a80f-4cad-b2a5-b297be04d53d, abstract = {{<p>Sulfite oxidase (SO) is an essential molybdoenzyme for humans, catalyzing the final step in the degradation of sulfur-containing amino acids and lipids, which is the oxidation of sulfite to sulfate. The catalytic site of SO consists of a molybdenum ion bound to the dithiolene sulfurs of one molybdopterin (MPT) molecule, carrying two oxygen ligands, and is further coordinated by the thiol sulfur of a conserved cysteine residue. We have exchanged four non-active site cysteines in the molybdenum cofactor (Moco) binding domain of human SO (SOMD) with serine using site-directed mutagenesis. This facilitated the specific replacement of the active site Cys207 with selenocysteine during protein expression in Escherichia coli. The sulfite oxidizing activity (k <sub>cat</sub>/K<sub>M</sub>) of SeSOMD<sub>4Ser</sub> was increased at least 1.5-fold, and the pH optimum was shifted to a more acidic value compared to those of SOMD<sub>4Ser</sub> and SOMD<sub>4Cys(wt)</sub>. X-ray absorption spectroscopy revealed a Mo<sup>VI</sup>-Se bond length of 2.51 Å, likely caused by the specific binding of Sec207 to the molybdenum, and otherwise rather similar square-pyramidal S/Se(Cys)O<sub>2</sub>Mo<sup>VI</sup>S <sub>2</sub>(MPT) site structures in the three constructs. The low-pH form of the Mo(V) electron paramagnetic resonance (EPR) signal of SeSOMD<sub>4Ser</sub> was altered compared to those of SOMD<sub>4Ser</sub> and SOMD <sub>4Cys(wt)</sub>, with g<sub>1</sub> in particular shifted to a lower magnetic field, due to the Se ligation at the molybdenum. In contrast, the Mo(V) EPR signal of the high-pH form was unchanged. The substantially stronger effect of substituting selenocysteine for cysteine at low pH as compared to high pH is most likely due to the decreased covalency of the Mo-Se bond.</p>}}, author = {{Reschke, Stefan and Niks, Dimitri and Wilson, Heather and Sigfridsson, Kajsa G.V. and Haumann, Michael and Rajagopalan, K. V. and Hille, Russ and Leimkühler, Silke}}, issn = {{0006-2960}}, language = {{eng}}, month = {{11}}, number = {{46}}, pages = {{8295--8303}}, publisher = {{The American Chemical Society (ACS)}}, series = {{Biochemistry}}, title = {{Effect of exchange of the cysteine molybdenum ligand with selenocysteine on the structure and function of the active site in human sulfite oxidase}}, url = {{http://dx.doi.org/10.1021/bi4008512}}, doi = {{10.1021/bi4008512}}, volume = {{52}}, year = {{2013}}, }