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Functional expression and spectroscopic studies of Desulfovibrio vulgaris sulfite reductase.
202
Abstracts
Bl47
FUNCTIONAL EXPRESSION AND SPECTROSCOPIC STUDIES OF VULGARIS SULFITE REDUCTASE.
DESULFOVIBRIO A. Soriano, T. Evan’s Laboratory
and J. A. Cowan of Chemistry,
The Ohio State University, Columbus,
Ohio 43220, USA.
The assimilatory sulfite reductase (Sin) from the sulfate-reducing bacterium vulgaris (Hildenborough) [Mr - 23,500 Da] catalyzes the &electron reduction of SOs2- to S*-. It contains the coupled siroheme-[Fe&l prosthetic center common to this class of enzyme [1,21. By virtue of its size, solubility, and accessibility to direct physicochemical measurements (ID and 2D NMR, electrochemistry, among others), SiR represents an outstanding model system for understanding the structural basis of recognition, bond activation, and catalytic redox chemistry of inorganic anions and gaseous substrates. Desulfovibrio
Hind111
Sal1
Hind11
pDSK519IATSiR(SE)
To facilitate detailed structure/function studies on SiR, the SiR gene has been overexpressed by utilizing a triparental conjugation method that allowed transformation of a high copy number broad-host-range plasmid pDSK519 into the native Desulfovibrio source to give an expression vehicle that provided a greater than 50-fold increase in enzyme production relative to the native strain [31. The recombinant enzyme has been characterized by standard biochemical methods, nuclear magnetic resonance, electronic absorption, circular dichroism, and activity measurements. It cannot be distinguished from the native enzyme on the basis of spectroscopic characterization or enzyme activity. With larger quantities of enzyme in hand, high field lH NMR studies have revealed a previously undetected broad resonance -40 ppm in addition to the other paramagnetic resonances which have already been reported [4]. Preliminary NOE experiments suggest connectivities between some of the paramagnetic resonances. Electronic absorption spectra of oxidized and reduced enzyme, with and without added ligands, show marked variations from those exibited by E. coli sulfite and spinach nitrite reductases. Both the assimilatory sulfite reductase and the larger dissimilatory enzyme show distinct spectral details with o-donor and n-acceptor ligands. 1. 2. 3.
4.
B. H. Huynh, L. Kang, D. V. DerVartanian, H. D. Peck, J. LeGall 1. Biol. Chern 259 15373 (1984). Jian Tan, L. Helms, R. I’. Swenson, J. A. Cowan Biochemistry 31 9900 (1991). B. J. Rapp, J. D.Wall PYOC. Natl. Acad. Sci. USA 84 9128 (1987). J. A. Cowan, M. Sola Inorganic Chemistry 29 2176 (1990).
Abstracts
Bl47
FUNCTIONAL EXPRESSION AND SPECTROSCOPIC STUDIES OF VULGARIS SULFITE REDUCTASE.
DESULFOVIBRIO A. Soriano, T. Evan’s Laboratory
and J. A. Cowan of Chemistry,
The Ohio State University, Columbus,
Ohio 43220, USA.
The assimilatory sulfite reductase (Sin) from the sulfate-reducing bacterium vulgaris (Hildenborough) [Mr - 23,500 Da] catalyzes the &electron reduction of SOs2- to S*-. It contains the coupled siroheme-[Fe&l prosthetic center common to this class of enzyme [1,21. By virtue of its size, solubility, and accessibility to direct physicochemical measurements (ID and 2D NMR, electrochemistry, among others), SiR represents an outstanding model system for understanding the structural basis of recognition, bond activation, and catalytic redox chemistry of inorganic anions and gaseous substrates. Desulfovibrio
Hind111
Sal1
Hind11
pDSK519IATSiR(SE)
To facilitate detailed structure/function studies on SiR, the SiR gene has been overexpressed by utilizing a triparental conjugation method that allowed transformation of a high copy number broad-host-range plasmid pDSK519 into the native Desulfovibrio source to give an expression vehicle that provided a greater than 50-fold increase in enzyme production relative to the native strain [31. The recombinant enzyme has been characterized by standard biochemical methods, nuclear magnetic resonance, electronic absorption, circular dichroism, and activity measurements. It cannot be distinguished from the native enzyme on the basis of spectroscopic characterization or enzyme activity. With larger quantities of enzyme in hand, high field lH NMR studies have revealed a previously undetected broad resonance -40 ppm in addition to the other paramagnetic resonances which have already been reported [4]. Preliminary NOE experiments suggest connectivities between some of the paramagnetic resonances. Electronic absorption spectra of oxidized and reduced enzyme, with and without added ligands, show marked variations from those exibited by E. coli sulfite and spinach nitrite reductases. Both the assimilatory sulfite reductase and the larger dissimilatory enzyme show distinct spectral details with o-donor and n-acceptor ligands. 1. 2. 3.
4.
B. H. Huynh, L. Kang, D. V. DerVartanian, H. D. Peck, J. LeGall 1. Biol. Chern 259 15373 (1984). Jian Tan, L. Helms, R. I’. Swenson, J. A. Cowan Biochemistry 31 9900 (1991). B. J. Rapp, J. D.Wall PYOC. Natl. Acad. Sci. USA 84 9128 (1987). J. A. Cowan, M. Sola Inorganic Chemistry 29 2176 (1990).