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J. Biol. Chem., Vol. 282, Issue 39, 28455-28464, September 28, 2007

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Ca²⁺-binding and Ca²⁺-independent Respiratory NADH and NADPH Dehydrogenases of Arabidopsis thaliana^*

From the Department of Cell and Organism Biology, Lund University, Sölvegatan 35B, SE-223 62 Lund, Sweden

Type II NAD(P)H:quinone oxidoreductases are single polypeptide proteins widespread in the living world. They bypass the first site of respiratory energy conservation, constituted by the type I NADH dehydrogenases. To investigate substrate specificities and Ca²⁺ binding properties of seven predicted type II NAD(P)H dehydrogenases of Arabidopsis thaliana we have produced them as T7-tagged fusion proteins in Escherichia coli. The NDB1 and NDB2 enzymes were found to bind Ca²⁺, and a single amino acid substitution in the EF hand motif of NDB1 abolished the Ca²⁺ binding. NDB2 and NDB4 functionally complemented an E. coli mutant deficient in endogenous type I and type II NADH dehydrogenases. This demonstrates that these two plant enzymes can substitute for the NADH dehydrogenases in the bacterial respiratory chain. Three NDB-type enzymes displayed distinct catalytic profiles with substrate specificities and Ca²⁺ stimulation being considerably affected by changes in pH and substrate concentrations. Under physiologically relevant conditions, the NDB1 fusion protein acted as a Ca²⁺-dependent NADPH dehydrogenase. NDB2 and NDB4 fusion proteins were NADH-specific, and NDB2 was stimulated by Ca²⁺. The observed activity profiles of the NDB-type enzymes provide a fundament for understanding the mitochondrial system for direct oxidation of cytosolic NAD(P)H in plants. Our findings also suggest different modes of regulation and metabolic roles for the analyzed A. thaliana enzymes.

Received for publication, June 6, 2007 , and in revised form, July 26, 2007.

^* This investigation was supported by The Swedish Natural Science 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ To whom correspondence should be addressed. Fax: 46-46-2224113; E-mail: Allan.Rasmusson{at}cob.lu.se.

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