Microsomal Electron Transfer in Higher Plants: Cloning and Heterologous Expression of NADH-Cytochrome b₅ Reductase from Arabidopsis

Masako Fukuchi-Mizutani^*, Masaharu Mizutani, Yoshikazu Tanaka, Takaaki Kusumi, and Daisaku Ohta

Institute for Fundamental Research, Suntory Limited, 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-0024, Japan (M.F.-M., Y.T., T.K.); Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan (M.M.); and College of Agriculture, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan (D.O.)

AtCBR, a cDNA encoding NADH-cytochrome (Cyt) b₅ reductase, and AtB5-A and AtB5-B, two cDNAs encoding Cyt b₅, were isolated from Arabidopsis. The primary structure deduced from the AtCBR cDNA was 40% identical to those of the NADH-Cyt b₅ reductases of yeast and mammals. A recombinant AtCBR protein prepared using a baculovirus system exhibited typical spectral properties of NADH-Cyt b₅ reductase and was used to study its electron-transfer activity. The recombinant NADH-Cyt b₅ reductase was functionally active and displayed strict specificity to NADH for the reduction of a recombinant Cyt b₅ (AtB5-A), whereas no Cyt b₅ reduction was observed when NADPH was used as the electron donor. Conversely, a recombinant NADPH-Cyt P450 reductase of Arabidopsis was able to reduce Cyt b₅ with NADPH but not with NADH. To our knowledge, this is the first evidence in higher plants that both NADH-Cyt b₅ reductase and NADPH-Cyt P450 reductase can reduce Cyt b₅ and have clear specificities in terms of the electron donor, NADH or NADPH, respectively. This substrate specificity of the two reductases is discussed in relation to the NADH- and NADPH-dependent activities of microsomal fatty acid desaturases.

Plant Physiol. (1999) 119: 353-362
Copyright Clearance Center:   0032-0889/99/119//10
© 1999 American Society of Plant Physiologists

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