Microsomal electron transfer in higher plants:: Cloning and heterologous expression of NADH-cytochrome b5 reductase from arabidopsis

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(5) reductases 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-Cyt b(5) reductase was functionally active and displayed strict specificity to NADH for the reduction of ;a recombinant Cyt b(5) (AtB5-A), whereas no Cyt b(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 Cyt b(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.