Microsomal Electron Transfer in Higher Plants: Cloning and Heterologous Expression of NADH-Cytochromeb5Reductase from Arabidopsis

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Abstract
AtCBR, a cDNA encoding NADH-cytochrome (Cyt)b 5 reductase, and AtB5-A and AtB5-B, two cDNAs encoding Cyt b 5, were isolated from Arabidopsis. The primary structure deduced from the AtCBR cDNA was 40% identical to those of the NADH-Cyt b 5reductases of yeast and mammals. A recombinant AtCBR protein prepared using a baculovirus system exhibited typical spectral properties of NADH-Cyt b 5 reductase and was used to study its electron-transfer activity. The recombinant NADH-Cytb 5 reductase was functionally active and displayed strict specificity to NADH for the reduction of a recombinant Cyt b 5 (AtB5-A), whereas no Cytb 5 reduction was observed when NADPH was used as the electron donor. Conversely, a recombinant NADPH-Cyt P450 reductase of Arabidopsis was able to reduce Cytb 5 with NADPH but not with NADH. To our knowledge, this is the first evidence in higher plants that both NADH-Cyt b 5 reductase and NADPH-Cyt P450 reductase can reduce Cyt b 5 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.

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