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Spectroscopic and kinetic studies of Nor1, a cytochrome P450 nitric oxide reductase from the fungal pathogen Histoplasma capsulatum |
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| Authors: | Chao Lily Y Rine Jasper Marletta Michael A |
| Institution: | a Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA b Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA c Department of Chemistry, University of California, Berkeley, CA, USA d Division of Physical Biosciences, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA, USA e California Institute of Quantitative Biosciences, University of California, Berkeley, CA, USA |
| Abstract: | The fungal respiratory pathogen Histoplasma capsulatum evades the innate immune response and colonizes macrophages during infection. Although macrophage production of the antimicrobial effector nitric oxide (NO) restricts H. capsulatum growth, the pathogen is able to establish a persistent infection. H. capsulatum contains a P450 nitric oxide reductase homologue (NOR1) that may be important for detoxifying NO during infection. To characterize the activity of this putative P450 enzyme, a 404 amino acid fragment of Nor1p was expressed in Escherichia coli and purified to homogeneity. Spectral characterization of Nor1p indicated that it was similar to other fungal P450 nitric oxide reductases. Nor1p catalyzed the reduction of NO to N2O using NADH as the direct reductant. The KM for NO was determined to be 20 μM and the kcat to be 5000 min−1. Together, these results provide evidence for a protective role of a P450 nitric oxide reductase against macrophage-derived NO. |
| Keywords: | Histoplasma capsulatum Nitric oxide Nitric oxide detoxification Cytochrome P450 Cytochrome P450 nitric oxide reductase Flavohemoglobin |
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