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Preliminary X-ray diffraction studies on the hemoprotein subunit of Escherichia coli sulfite reductase
J. Mol. Biol.
(1982) 154, 179-180
Preliminary X-ray Diffraction Subunit of Escherichk
Studies on the Hemoprotein coli Sulfite Reductase
Crystals of the hemoprotein subunit of Escherichia coli sulfite reductase have been grown and analyzed by X-ray diffraction. The crystals grow as needles with cell dimensions of a = 69.7 A, b = 77.4 A, c = 88+3 A, space group P2,2,2,, and one monomer per asymmetric unit.
NADPH-sulfite reductase (EC1.8.1.2) catalyzes the six-electron reduction of sulfite to sulfide using NADPH as the electron donor. This reaction is involved in the biosynthesis of cysteine in plants and bacteria. Excherichia coli sulfite reductase is a multi-center enzyme containing four different prosthetic groups: FAD, FMN, siroheme and a 4-iron-4-sulfur center (Siegel et aE., 1973). The electron flow from XADPH to sulfite follows the sequence: NADPH-FAD-FMN-heme-sulfite. with the role of the iron-sulfur center unclear. This enzyme may be a soluble model for membrane-bound electron transport chains, and so we wish to determine the steric relationships of the various electron centers. The enzyme isolated from E. co2i has a molecular weight of 670,000. It is composed of two different polypeptide chains: B. which binds the FAD and FMN prosthetic groups and is termed the flavoprotein, and 8, which binds the siroheme and the iron-sulfur center and is termed the hemoprotein. The holoenzyme has the structure “s/l4 and can be dissociated into the flavoprotein and hemoprotein components by DEAE-cellulose chromat,ography in 5 M-Urea (Siegel & Davis, 1974). In solution the separated flavoprotein forms an octamer and is capable of accepting electrons from NADPH. The hemoprobein is a monomer and can reduce sulfit,e using reduced methyl viologen as the electron donor. The fact that the hemoprotein contains both an iron-sulfur center and the novel siroheme makes it an interesting candidate for a detailed structural study. In this letter we describe t,he crystallization monomer of the hemoprotein and its preliminary characterization by X-ray diffraction. (‘rystals of the hemoprotein were grown at room temperature and at 4°C from solutions initially containing 20 mg protein/ml in 0.05 M-potassium phosphate buffer at pH 7.8. A solution of 40% (w/v) polyethylene glycol 6000 in the same buffer was added to reach a final concentratjion of about 1696 polyethylene glycol. The crystals have a needle-like morphology and are always accompanied by the formation of a flocculent precipitate that does not appear to interfere with crystal growth. The crystals will grow over a pH range of 7.5 to 95 and at phosphate concentrations of 0.005 M to 0.1 M. Precession X-ray diffract’ion photographs show a diffraction pattern consistent with space group P2,2,2, with unit cell dimensions of a = 69.7 A, b = 77.4& and c = 88.8 A. Using a molecular weight of 55,000 for the hemoprotein monomer 179 0022-2836/82/010179-02
$02.00/O
0 1982 Academic Press Inc. (London)
Ltd.
I). R. MrRE:R
180
ANI)
1). (‘. RICHARDSON
(Siegel Hr Davis, 1074) and assuming one monomer per asymmetric unit. gives a cell volume per dalton of 2.2 a3/dalton, well within the normal range of values (Matthews. 1968). Diffraction with Bragg spacings out to 2.3 a has bean observed on Laue photographs. ‘Vl’ork is proceeding on the X-ray crystallography of the hemoprotein with the eventual goal of obtaining the three-dimensional structure at high resolution. The authors are indebted to Dr Lewis M. Siegel for supplying the enzyme. The work supported by National Institutes of Health grant GM15000. One of us (D.E.M.) supported by Public Health Services Research Service Award GM07184. Depart.ment of Biochemistry Duke University Durham. NC 17710. I’.S.A. Received
1 September
was was
DI‘NCAN E. MCRIW DAVII) (‘. RICHAKIHOK
1981
REFERENCES Matthews. B. IV. (1968). J. iI1ol. Riol. 33, 491-497. Siegel. I,. M. & Davis. I’. S. (1974). J. Riol. Chem. 249, 158771598. Siegel. L. M., Murphy. M. ,J. & Kamin, H. (1973). J. Riol. Chem. 248, 251-264.
Edited
by A. Klug
(1982) 154, 179-180
Preliminary X-ray Diffraction Subunit of Escherichk
Studies on the Hemoprotein coli Sulfite Reductase
Crystals of the hemoprotein subunit of Escherichia coli sulfite reductase have been grown and analyzed by X-ray diffraction. The crystals grow as needles with cell dimensions of a = 69.7 A, b = 77.4 A, c = 88+3 A, space group P2,2,2,, and one monomer per asymmetric unit.
NADPH-sulfite reductase (EC1.8.1.2) catalyzes the six-electron reduction of sulfite to sulfide using NADPH as the electron donor. This reaction is involved in the biosynthesis of cysteine in plants and bacteria. Excherichia coli sulfite reductase is a multi-center enzyme containing four different prosthetic groups: FAD, FMN, siroheme and a 4-iron-4-sulfur center (Siegel et aE., 1973). The electron flow from XADPH to sulfite follows the sequence: NADPH-FAD-FMN-heme-sulfite. with the role of the iron-sulfur center unclear. This enzyme may be a soluble model for membrane-bound electron transport chains, and so we wish to determine the steric relationships of the various electron centers. The enzyme isolated from E. co2i has a molecular weight of 670,000. It is composed of two different polypeptide chains: B. which binds the FAD and FMN prosthetic groups and is termed the flavoprotein, and 8, which binds the siroheme and the iron-sulfur center and is termed the hemoprotein. The holoenzyme has the structure “s/l4 and can be dissociated into the flavoprotein and hemoprotein components by DEAE-cellulose chromat,ography in 5 M-Urea (Siegel & Davis, 1974). In solution the separated flavoprotein forms an octamer and is capable of accepting electrons from NADPH. The hemoprobein is a monomer and can reduce sulfit,e using reduced methyl viologen as the electron donor. The fact that the hemoprotein contains both an iron-sulfur center and the novel siroheme makes it an interesting candidate for a detailed structural study. In this letter we describe t,he crystallization monomer of the hemoprotein and its preliminary characterization by X-ray diffraction. (‘rystals of the hemoprotein were grown at room temperature and at 4°C from solutions initially containing 20 mg protein/ml in 0.05 M-potassium phosphate buffer at pH 7.8. A solution of 40% (w/v) polyethylene glycol 6000 in the same buffer was added to reach a final concentratjion of about 1696 polyethylene glycol. The crystals have a needle-like morphology and are always accompanied by the formation of a flocculent precipitate that does not appear to interfere with crystal growth. The crystals will grow over a pH range of 7.5 to 95 and at phosphate concentrations of 0.005 M to 0.1 M. Precession X-ray diffract’ion photographs show a diffraction pattern consistent with space group P2,2,2, with unit cell dimensions of a = 69.7 A, b = 77.4& and c = 88.8 A. Using a molecular weight of 55,000 for the hemoprotein monomer 179 0022-2836/82/010179-02
$02.00/O
0 1982 Academic Press Inc. (London)
Ltd.
I). R. MrRE:R
180
ANI)
1). (‘. RICHARDSON
(Siegel Hr Davis, 1074) and assuming one monomer per asymmetric unit. gives a cell volume per dalton of 2.2 a3/dalton, well within the normal range of values (Matthews. 1968). Diffraction with Bragg spacings out to 2.3 a has bean observed on Laue photographs. ‘Vl’ork is proceeding on the X-ray crystallography of the hemoprotein with the eventual goal of obtaining the three-dimensional structure at high resolution. The authors are indebted to Dr Lewis M. Siegel for supplying the enzyme. The work supported by National Institutes of Health grant GM15000. One of us (D.E.M.) supported by Public Health Services Research Service Award GM07184. Depart.ment of Biochemistry Duke University Durham. NC 17710. I’.S.A. Received
1 September
was was
DI‘NCAN E. MCRIW DAVII) (‘. RICHAKIHOK
1981
REFERENCES Matthews. B. IV. (1968). J. iI1ol. Riol. 33, 491-497. Siegel. I,. M. & Davis. I’. S. (1974). J. Riol. Chem. 249, 158771598. Siegel. L. M., Murphy. M. ,J. & Kamin, H. (1973). J. Riol. Chem. 248, 251-264.
Edited
by A. Klug