Magneto-optical studies of RirA and cytochrome cd1
Rhizobia are members of the Gram-negative α-proteobacteria. Rhizobium leguminosarum controls genes involved in iron uptake through repression under conditions of iron sufficiency. This part of iron regulation is co-ordinated by the dimeric rhizobial iron regulator, RirA. The sequence of RirA contains four cysteines in close proximity, three of which have been shown to be essential to the regulatory functions of RirA and have been proposed as the binding site of an iron-sulfur cluster. RirA has been overexpressed, purified and reconstituted with an ironsulphur cluster. EPR spectroscopy combined with iron and sulphide assays suggests that the protein binds a [4Fe-4S] or [3Fe-4S] cluster per monomer. Cytochrome cd1 is a homodimeric nitrite reductase found in denitrifying bacteria. Each monomer binds a heme c that transfers electrons to the structurally unique heme d1 at the active site. It is shown that the inactive as-prepared form of cd1 from Paracoccus pantotrophus is activated by pre-exposure to the substrate NO2 −. Although oxidised cd1 from Pseudomonas aeruginosa does not require similar activation, it undergoes the same reaction with NO2 − to produce a novel form of nitrosyl-heme d1. The NO derivative of Pseudomonas aeruginosa cd1 was prepared and characterised using variable-temperature variable-field (VTVF) Magnetic Circular Dichroism (MCD) spectroscopy. The novel heme d1 nitrosyl has thus been identified as an unprecedented FeIII-NO• species. In absorption spectra, this form of nitrosyl-heme d1 is indistinguishable from the low-spin FeIII state. But simulation of EPR spectra of the oxidised cd1s has allowed quantitation of the two hemes and shows that there are no populations of the FeIII-NO• product-bound active site heme.
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