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J. Biol. Chem., Vol. 282, Issue 17, 12517-12526, April 27, 2007

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Biochemical Studies of Klebsiella pneumoniae NifL Reduction Using Reconstituted Partial Anaerobic Respiratory Chains of Wolinella succinogenes^*

Robert Thummer, Oliver Klimmek, and Ruth A. Schmitz¹

From the Institut für Allgemeine Mikrobiologie, Christian-Albrechts Universität zu Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany and Institut für Molekulare Biowissenschaften, Johann Wolfgang Goethe-Universität, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany

In the diazotroph Klebsiella pneumoniae the flavoprotein NifL inhibits the activity of the nif-specific transcriptional activator NifA in response to molecular oxygen and combined nitrogen. Sequestration of reduced NifL to the cytoplasmic membrane under anaerobic and nitrogen-limited conditions impairs inhibition of cytoplasmic NifA by NifL. To analyze whether NifL is reduced by electrons directly derived from the reduced menaquinone pool, we studied NifL reduction using artificial membrane systems containing purified components of the anaerobic respiratory chain of Wolinella succinogenes. In this in vitro assay using proteoliposomes containing purified formate dehydrogenase and purified menaquinone (MK₆) or 8-methylmenaquinone (MMK₆) from W. succinogenes, reduction of purified NifL was achieved by formate oxidation. Furthermore, the respective reduction rates, which were determined using equal amounts of NifL, have been shown to be directly dependent on the concentration of both formate dehydrogenase and menaquinones incorporated into the proteoliposomes, demonstrating a direct electron transfer from menaquinone to NifL. When purified hydrogenase and MK₆ from W. succinogenes were inserted into the proteoliposomes, NifL was reduced with nearly the same rate by hydrogen oxidation. In both cases reduced NifL was found to be highly associated to the proteoliposomes, which is in accordance with our previous findings in vivo. On the bases of these experiments, we propose that the redox state of the menaquinone pool is the redox signal for nif regulation in K. pneumoniae by directly transferring electrons onto NifL under anaerobic conditions.

Received for publication, October 18, 2006 , and in revised form, February 15, 2007.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SCHM1052/8-1 and the Fonds der Chemischen Industrie. 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.

¹ To whom correspondence should be addressed. Tel.: 49-431-8804334; Fax: 49-431-8802194; E-mail: rschmitz{at}ifam.uni-kiel.de.

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