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J. Biol. Chem., Vol. 279, Issue 31, 32212-32218, July 30, 2004

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Tuning a Nitrate Reductase for Function

THE FIRST SPECTROPOTENTIOMETRIC CHARACTERIZATION OF A BACTERIAL ASSIMILATORY NITRATE REDUCTASE REVEALS NOVEL REDOX PROPERTIES^*

Brian J. N. Jepson, Lee J. Anderson, Luis M. Rubio¶, Clare J. Taylor, Clive S. Butler||, Enrique Flores¶, Antonia Herrero¶, Julea N. Butt, and David J. Richardson**

From the Centre for Metalloprotein Spectroscopy and Biology, Schools of Biological Sciences and Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom, the ^¶Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas-Universidad de Sevilla, E-41092 Seville, Spain, and the ^||School of Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne NE2 4HH, United Kingdom

Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate reductases. In the present study the ferredoxin-dependent nitrate reductase NarB of the cyanobacterium Synechococcus sp. PCC 7942 was analyzed by spectropotentiometry and protein film voltammetry. Metal and acid-labile sulfide analysis revealed nearest integer values of 4:4:1 (iron/sulfur/molybdenum)/molecule of NarB. Analysis of dithionite-reduced enzyme by low temperature EPR revealed at 10 K the presence of a signal that is characteristic of a [4Fe-4S]¹⁺ cluster. EPR-monitored potentiometric titration of NarB revealed that this cluster titrated as an n = 1 Nernstian component with a midpoint redox potential (E_m) of –190 mV. EPR spectra collected at 60 K revealed a Mo(V) signal termed "very high g" with g_av = 2.0047 in air-oxidized enzyme that accounted for only 10–20% of the total molybdenum. This signal disappeared upon reduction with dithionite, and a new "high g" species (g_av = 1.9897) was observed. In potentiometric titrations the high g Mo(V) signal developed over the potential range of –100 to –350 mV (E_m Mo^6+/5+ = –150 mV), and when fully developed, it accounted for 1 mol of Mo(V)/mol of enzyme. Protein film voltammetry of NarB revealed that activity is turned on at potentials below –200 mV, where the cofactors are predominantly [4Fe-4S]¹⁺ and Mo⁵⁺. The data suggests that during the catalytic cycle nitrate will bind to the Mo⁵⁺ state of NarB in which the enzyme is minimally two-electron-reduced. Comparison of the spectral properties of NarB with those of the membrane-bound and periplasmic respiratory nitrate reductases reveals that it is closely related to the periplasmic enzyme, but the potential of the molybdenum center of NarB is tuned to operate at lower potentials, consistent with the coupling of NarB to low potential ferredoxins in the cell cytoplasm.

Received for publication, March 9, 2004 , and in revised form, May 24, 2004.

^* This work was supported by Grants P13842 and B17233 from the United Kingdom Biotechnology and Biological Sciences Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed. Tel.: 44-1603-593250; Fax: 44-1603-592250; E-mail: d.richardson{at}uea.ac.uk.

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