Functional analysis and subcellular location of two flavohemoglobins from Aspergillus oryzae

Multiple flavohemoglobin (FHb) homolog genes are found in the genomes of eukaryotic microorganisms, but their functions remain unknown. In this study, two distinct types of FHbs (predictive cytosolic FHb1 and predictive mitochondrial FHb2) from the fungus Aspergillus oryzae were investigated to elucidate the physiological roles of these FHbs. The fhb1 gene responded to external nitric oxide (NO) stress at the transcriptional level, whereas the fhb2 gene did not. Disrupting fhb1 increased cell hypersensitivity to NO stress, whereas deficiency of the fhb2 gene had no effect on phenotype compared to the wild-type strain. By fusing GFP protein to FHbs, we determined that FHb1 and FHb2 are located in the cytosol and mitochondria, respectively. In the wild-type strain, the transcriptional level of the fhb2 gene was too low to be detected, but its expression was detectable in the NirK (mitochondrial copper-containing dissimilatory nitrite reductase) overexpression strain (AoHnirK), which showed a significantly higher denitrification capability than that shown by the wild-type strain. The induction of the fhb2 gene in the AoHnirK strain may be due to the abundance of NO produced by overexpressed NirK in the mitochondria. These results suggest that FHb1 and FHb2 may play a role in protecting cells from external and internal NO stress, respectively.