Aspartate 205 in the catalytic domain of naphthalene dioxygenase is essential for activity

The naphthalene dioxygenase enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4, The crystal structure of naphthalene dioxygenase (B, Kauppi, K, Lee, E, Car redano, R, E, Parales, D, T, Gibson, H. Eklund, and S. Ramaswamy, Structure 6:571-586, 1998) indicates that aspartate 205 may provide the most direct route of electron transfer between the Rieske [2Fe-2S] center of one alpha subunit and mononuclear iron in the adjacent alpha subunit, In this study, we constructed four site-directed mutations that changed aspartate 205 to a lanine, glutamate, asparagine, or glutamine to test whether this residue is essential for naphthalene dioxygenase activity. The mutant proteins were v ery inefficient in oxidizing naphthalene to cis-naphthalene dihydrodiol, an d oxygen uptake in the presence of naphthalene was below detectable levels. The purified mutant protein with glutamine in place of aspartate 205 had i dentical spectral properties to wild-type naphthalene dioxygenase and was r educed by NADH in the presence of catalytic amounts of ferredoxin(NAP) and reductase(NAP) Benzene, an effective uncoupler of oxygen consumption in pur ified naphthalene dioxygenase, did not elicit oxygen uptake by the mutant p rotein. These results indicate that electron transfer from NADH to the Ries ke center in the mutant oxygenase is intact, a finding consistent with the proposal that aspartate 205 is a necessary residue in the major pathway of electron transfer to mononuclear iron at the active site.