Nitrite reductase (NiR) catalyzes the reduction of nitrite to nitric oxide in the denitrification stage of the nitrogen cycle. To study the structure and function of this noncoupled dinuclear copper enzyme, models were designed by adding a second copper binding site on the surface of the mononuclear copper protein azurin. The existing copper-binding site in azurin is an electron transfer (T1) site and it is ligated by two histidines, a cysteine and a methionine residue similar to the T1 site of NiR. This T1 copper site was maintained in azurin whereas an additional copper center, a catalytic type 2 (T2) copper site similar to the T2 site of NiR was designed. The designed T2 copper site in azurin models the spectroscopic and functional aspects of NiR. The reactivity, UV-Vis absorption and EPR spectra of the NiR-azurin models showed similarities to that of native NiR but were less active. We have further examined the NiR activity of our models using the Griess assay. Also, reduction and re-oxidation assays of the T1 center in the NiR-azurin models were determined and were compared with variants with modified T1 copper center redox potentials. This yielded insights into the order of the reaction between the reducing agent, protein, and the substrate in our models. Recent results will be discussed.

Nitrite reduction activity of azurin variants

249th National Meeting (2015)

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