Assignment of haem ligands and detection of electronic absorption bands ofmolybdenum in the di-haem periplasmic nitrate reductase of Paracoccus pantotrophus

The periplasmic nitrate reductase (NAP) from Paracoccus pantotrophus is a s oluble two-subunit enzyme (NapAB) that binds two c-type haems, a [4Fe-4S] c luster and a bis-molybdopterin guanine dinucleotide cofactor that catalyses the reduction of nitrate to nitrite, In the present,work the NapAB complex has been studied by magneto-optical spectroscopy to probe co-ordination of both the NapB haems and the NapA active site Mo. The absorption spectrum o f the NapAB complex is dominated by features from the NapB c-type cytochrom es, Using a combination of electron paramagnetic resonance spectroscopy and magnetic circular dichroism it was demonstrated that both haems are low-sp in with bis-histidine axial ligation, In addition, a window between 600 and 800 nm was identified in which weak absorption features that may arise fro m Mo could be detected. The low-temperature MCD spectrum shows oppositely s igned bands in this region (peak 648 nm, trough 714 nm) which have been ass igned to S-to-Mo(V) charge transfer transitions. (C) 2001 Published by Else vier Science B,V, on behalf of the Federation of European Biochemical Socie ties.