Purification and cDNA cloning of nitric oxide reductase cytochrome P450nor(CYP55A4) from Trichosporon cutaneum

Cytochrome P450nor is involved in fungal denitrification as nitric oxide (N O) reductase. Although the heme protein has been known to occur in restrict ed species of fungi that belong to ascomycotina, we have previously suggest ed that it would also occur in the yeast Trichosporon cutaneum, which is ph ylogenetically far from those P450nor-producing ascomycetous fungi. Here we isolated and characterized the heme protein from the basidiomycetous yeast T. cutaneum. P450nor of the yeast (TcP450nor) exhibited properties in term s of catalysis, absorption spectrum and molecular mass that are almost iden tical to those of its counterparts in ascomycetous fungi. We also isolated and sequenced its cDNA. The predicted primary structure of TcP450nor showed high sequence identities (around 65%) to those of other P450nors, indicati ng that they belong to the same family. TcP450nor protein cofractionated wi th cytochrome c oxidase by subcellular fractionation and its predicted prim ary structure contained an extension on its amino terminus that is characte ristic of a mitochondrial-targeting signal, indicating that it is a mitocho ndrial protein like some of the isoforms of other fungi. On the other hand, TcP450nor was unique in that inducers such as nitrate, nitrite, or NO were not required for its production in the cells. The occurrence of P450nor ac ross the subdivisions of eumycota suggests that P450nor and denitrification are distributed more universally among fungi than was previously thought.