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Crystallographic analysis of sulfite reduction mediated by a Siroheme-Fe4S4 coupled redox system in the catalytic subunit of E. coli sulfite reductase
STRUCTURE/FUNCTION
B134
Crystallographic Analysis of Sulfite Reduction Mediated by a Siroheme-Fe& Coupled Redox System in The Catalytic Subunit of E. coli Sulfite Reductase
Brian R. Crane O, Duncan E. McReeO, Lewis M. Siegelb and Elizabeth D. Getzoff “Department of Molecular Biology, The Scripps Research Institiute, La Jolla California, 92097. bDepartments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710. Reduction of the inorganic substrates sulfite and nitrite are obligatory steps for the assimilation of sulfur and nitrogen into the biosphere. To gain further understanding of the enzymatic redox chemistry involved we are defining the high resolution crystallographic structure of the E.coli NADPH Sulfite Reductase Hemoprotein Subunit (SiRHP). When supplied with artificial electron donors, SiRHP will perform the concerted six-electron reduction of sulfite to sulfide, and nitrite to ammonia, without releasing any detectable intermediates [l]. The unique active site assembly of this 64,000 Da enzyme consists of a siroheme, (an unusual iron-tetrahydroporphyrin of the isobacteriochlorin family), coupled structurally and electronically to an Fe,& cluster via a putative cysteinate sulfur bridge [2]. There is much spectroscopic evidence &s well as preliminary crystallographic work in support of this model 13-51. The SiRHP crystals diffract to 1.7 A, however, the system has proven refractory to traditional heavy atom replacement methodology. In order to augment existing phasing information we have used tunable synchrotron radiation to accentuate wavelength-dependent anomalous scattering effects caused by the native iron atoms of the protein. This treatment resulted in considerably improved electron density maps. Elements of secondary structure and the direction of the polypeptide chain are now discernible. The 570 residues of the oxidized enzyme are currently being built into this density. 1. Siegel, L.M., Rueger, D.C., Barber, M.J., Krueger, R.J., Orme-Johnson, N.R. and Orme-Johnson, W.H. J. Biol. Chem., 257,6343-6350 (1982). 2. Ostrowski, J., Wu, J-Y, Ruegers, D.C., Miller, B.E., Siegel, L.M. and Kredich, N.M. J. Biol. Chem., 264, 15726-15737 (1989). 3. McRee, D.E., Richardson, D.C., Richardson, J.S. and Siegel, L.M. J. Biol. Chem., 261, 10277-10281(1986). 4. Christner, J. A., Janick, P. A., Siegel, L. M., and Miinck, E. J. Biol. Chem., 258, 11157-11164 (1983). 5. Janick, P. A., and Siegel, L.M. Biochemistry, 22, 504-515 (1983).
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B134
Crystallographic Analysis of Sulfite Reduction Mediated by a Siroheme-Fe& Coupled Redox System in The Catalytic Subunit of E. coli Sulfite Reductase
Brian R. Crane O, Duncan E. McReeO, Lewis M. Siegelb and Elizabeth D. Getzoff “Department of Molecular Biology, The Scripps Research Institiute, La Jolla California, 92097. bDepartments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710. Reduction of the inorganic substrates sulfite and nitrite are obligatory steps for the assimilation of sulfur and nitrogen into the biosphere. To gain further understanding of the enzymatic redox chemistry involved we are defining the high resolution crystallographic structure of the E.coli NADPH Sulfite Reductase Hemoprotein Subunit (SiRHP). When supplied with artificial electron donors, SiRHP will perform the concerted six-electron reduction of sulfite to sulfide, and nitrite to ammonia, without releasing any detectable intermediates [l]. The unique active site assembly of this 64,000 Da enzyme consists of a siroheme, (an unusual iron-tetrahydroporphyrin of the isobacteriochlorin family), coupled structurally and electronically to an Fe,& cluster via a putative cysteinate sulfur bridge [2]. There is much spectroscopic evidence &s well as preliminary crystallographic work in support of this model 13-51. The SiRHP crystals diffract to 1.7 A, however, the system has proven refractory to traditional heavy atom replacement methodology. In order to augment existing phasing information we have used tunable synchrotron radiation to accentuate wavelength-dependent anomalous scattering effects caused by the native iron atoms of the protein. This treatment resulted in considerably improved electron density maps. Elements of secondary structure and the direction of the polypeptide chain are now discernible. The 570 residues of the oxidized enzyme are currently being built into this density. 1. Siegel, L.M., Rueger, D.C., Barber, M.J., Krueger, R.J., Orme-Johnson, N.R. and Orme-Johnson, W.H. J. Biol. Chem., 257,6343-6350 (1982). 2. Ostrowski, J., Wu, J-Y, Ruegers, D.C., Miller, B.E., Siegel, L.M. and Kredich, N.M. J. Biol. Chem., 264, 15726-15737 (1989). 3. McRee, D.E., Richardson, D.C., Richardson, J.S. and Siegel, L.M. J. Biol. Chem., 261, 10277-10281(1986). 4. Christner, J. A., Janick, P. A., Siegel, L. M., and Miinck, E. J. Biol. Chem., 258, 11157-11164 (1983). 5. Janick, P. A., and Siegel, L.M. Biochemistry, 22, 504-515 (1983).
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