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The active site of the bacterial nitric oxide reductase from Paracoccus denitrificans contains a dinuclear centre comprising heme b 3 and non heme iron (Fe B ). These metal centres are shown to be at isopotential with midpoint reduction potentials of E m ≈ + 80 mV. The midpoint reduction potentials of the other two metal centres in the enzyme, heme c and heme b , are greater than the dinuclear centre suggesting that they act as an electron receiving/storage module. Reduction of the low-spin heme b causes structural changes at the dinuclear centre which allow access to substrate molecules. In the presence of the substrate analogue, CO, the midpoint reduction potential of heme b 3 is raised to a region similar to that of heme c and heme b . This leads us to suggest that reduction of the electron transfer hemes leads to an opening of the active site which allows substrate to bind and in turn raises the reduction potential of the active site such that electrons are only delivered to the active site following substrate binding.

The active site of nitric oxide reductase from Paracoccus denitrificans contains heme and non-heme iron and is evolutionarily related to heme-copper oxidases. The CO and NO dynamics in the active site were investigated using ultrafast transient absorption spectroscopy. We find that, upon photodissociation from the active site heme, 20% of the CO rebinds in 170 ps, suggesting that not all the CO transiently binds to the non-heme iron. The remaining 80% does not rebind within 4 ns and likely migrates out of the active site without transient binding to the non-heme iron. Rebinding of NO to ferrous heme takes place in approximately 13 ps. Our results reveal that heme-ligand recombination in this enzyme is considerably faster than in heme-copper oxidases and are consistent with a more confined configuration of the active site.