A Manganese(IV)/Iron(III) Cofactor in Chlamydia trachomatis Ribonucleotide Reductase

American Association for the Advancement of Science (AAAS) - Tập 316 Số 5828 - Trang 1188-1191 - 2007
Wei Jiang1,2, Danny Yun1,2, Lana Saleh1,2, Eric W. Barr1,2, Gang Xing1,2, Lee M. Hoffart1,2, Monique-Anne Maslak1,2, Carsten Krebs1,2, J. Martin Bollinger1,2
1Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA
2Department of Chemistry, Pennsylvania State University, University Park, PA 16802 USA

Tóm tắt

In a conventional class I ribonucleotide reductase (RNR), a diiron(II/II) cofactor in the R2 subunit reacts with oxygen to produce a diiron(III/IV) intermediate, which generates a stable tyrosyl radical (Y⚫). The Y⚫ reversibly oxidizes a cysteine residue in the R1 subunit to a cysteinyl radical (C⚫), which abstracts the 3′-hydrogen of the substrate to initiate its reduction. The RNR from Chlamydia trachomatis lacks the Y⚫, and it had been proposed that the diiron(III/IV) complex in R2 directly generates the C⚫ in R1. By enzyme activity measurements and spectroscopic methods, we show that this RNR actually uses a previously unknown stable manganese(IV)/iron(III) cofactor for radical initiation.

Từ khóa


Tài liệu tham khảo

P. Nordlund, P. Reichard, Annu. Rev. Biochem.75, 681 (2006).

10.1021/cr020421u

10.1126/science.271.5248.477

J. Stubbe, Curr. Opin. Struct. Biol.10, 731 (2000).

C. C. Lawrence, J. Stubbe, Curr. Opin. Chem. Biol.2, 650 (1998).

M. Fontecave, E. Mulliez, D. T. Logan, Prog. Nucleic Acid Res. Mol. Biol.72, 95 (2002).

J. Stubbe, Curr. Opin. Chem. Biol.7, 183 (2003).

C. Roshick, E. R. Iliffe-Lee, G. McClarty, J. Biol. Chem.275, 38111 (2000).

10.1126/science.1098419

N. Voevodskaya, F. Lendzian, A. Gräslund, Biochem. Biophys. Res. Commun.330, 1213 (2005).

N. Voevodskaya et al., Proc. Natl. Acad. Sci. U.S.A.103, 9850 (2006).

Materials and methods are available as supporting material on Science Online.

For example preparations from E. coli cultures grown in rich medium had the same iron content as preparations from iron-supplemented minimal medium (∼0.75 equiv; all metal equivalencies and activities are on a per monomer basis) but ∼10 times the activity [velocity ( v )/[R2] = 0.035 ± 0.01 s –1 compared with 0.003 ± 0.001 s –1 ]. Conversely R2 from rich medium to which the cell-permeative Fe II chelator 1 10-phenanthroline was added immediately before induction of overexpression emerged with much less iron (<0.05 equiv) but ∼70% of the activity ( v /[R2] = 0.025 ± 0.01 s –1 ) of the R2 from equivalent cultures lacking the chelator.

All metal equivalencies and activities for the homodimeric R2 protein are reported on a per monomer basis.

To reduce the residual R2 activity to this low level it was necessary also to dialyze the R1 used in the activity assay against EDTA.

The S = 1/2 complex exhibits a magnetically split Mössbauer spectrum at low temperature but the use of this higher temperature makes electronic relaxation fast compared with the 57 Fe nuclear precession frequency and collapses the spectrum into a quadrupole doublet.

M. Zheng, S. V. Khangulov, G. C. Dismukes, V. V. Barynin, Inorg. Chem.33, 382 (1994).

S. Sinnecker, F. Neese, W. Lubitz, J. Biol. Inorg. Chem.10, 231 (2005).

E. Münck, in Physical Methods in Bioinorganic Chemistry, L. Que Jr., Ed. (University Science Books, Sausalito, CA, 2000), pp. 287–319.

L. Thelander, B. Larsson, J. Hobbs, F. Eckstein, J. Biol. Chem.251, 1398 (1976).

R. Eliasson, E. Pontis, F. Eckstein, P. Reichard, J. Biol. Chem.269, 26116 (1994).

S. Salowe et al., Biochemistry32, 12749 (1993).

G. Behravan et al., Biochim. Biophys. Acta1264, 323 (1995).

J. Fritscher et al., J. Am. Chem. Soc.127, 7729 (2005).

B. M. Sjöberg, A. Gräslund, F. Eckstein, J. Biol. Chem.258, 8060 (1983).

10.1021/cr9400875

M. Lepoivre, F. Fieschi, J. Coves, L. Thelander, M. Fontecave, Biochem. Biophys. Res. Commun.179, 442 (1991).

O. Guittet, B. Roy, M. Lepoivre, Cell. Mol. Life Sci.55, 1054 (1999).

M. Fontecave, Cell. Mol. Life Sci.54, 684 (1998).

H. M. Hassan, L. W. Schrum, FEMS Microbiol. Rev.14, 315 (1994).

This work was supported by grants from NIH (GM55365 to J.M.B.) the Arnold and Mabel Beckman Foundation (Young Investigator Award to C.K.) and the Camille and Henry Dreyfus Foundation (Teacher Scholar Award to C.K.). The authors thank R. Stevens (University of California Berkeley) for generously providing Chlamydia trachomatis serovar D genomic DNA and J. Stubbe (Massachusetts Institute of Technology) for the kind gift of N 3 -ADP.

Thông tin
Thông tin xuất bản

American Association for the Advancement of Science (AAAS)

Tập 316 Số 5828

1188-1191

Thông tin tác giả