Evolution of copper-containing nitrite reductase

Copper-containing nitrite reductase (NiR) is a homotrimer of two cupredoxin domains and contains two spectroscopically distinct copper sitesa type-1 Cu that accepts electrons from a cupredoxin pseudoazurin and transfers them to a mononuclear type-2 Cu atom where NO2 is reduced to NO during denitrification. NiR has also been shown to reduce oxygen, for which the product is believed to be hydrogen peroxide (H2O2). Multicopper oxidases (MCOs) catalyze the reduction of oxygen to water and are homologous to NiR, containing multiple cupredoxin domains and a type-1 Cu site that transfers electrons to a trinuclear Cu active site, as opposed to a mononuclear active site of NiR. To investigate the evolution of NiR, methods of mutagenic library generation and high-throughput variant screening from E. coli colonies were developed. These methods allow for facile screening of 10 mutants for folding efficiency or substrate specificity. Initial proof of principle studies yielded several variants that oxidized the artificial substrate ο-dianisidine up to 8 times faster than wild type NiR, suggesting that this methodology has the potential to engineer NiR to acquire other reductase functions. A crystal structure was solved for a putative multicopper oxidase (MCO) and NiR homologue from Arthrobacter sp. (AMMCO) to 1.8 Å resolution. The overall folds of AMMCO and NiR are very similar (r.m.s.d. of 2.0 Å over 250 Cα atoms); Like NiR, AMMCO is a trimer with type-1 Cu sites in the N-terminal domain of each monomer; however, the active site of AMMCO contains trinuclear Cu site characteristic of MCOs instead of a the mononuclear type-2 Cu site found in NiR. Detailed structural analysis supports the theory that two-domain MCOs similar to AMMCO were intermediaries in iii the evolution of NiR and the more common three-domain MCOs. The physiological function of AMMCO remains uncertain, but genomic, crystallographic and functional analysis suggests that the enzyme is involved in metal regulation. AMMCO homologues are co-localized within multiple genomes with a homologue of the ZIP family of zinc transporters. The x-ray crystal structure shows that a distinct loop of the enzyme (residues 184-192) bound calcium in distorted octahedral fashion. Calcium may not be the physiological metal bound at this site due to the high concentration (>200 mM) of Ca required for crystallization. The AMMCO calcium binding loop was altered by sitedirected mutagenesis: Asp190Ala and Asp190Ala/Glu192Ala variants. These variants display altered electronic absorption spectra (shift in the 600 nm peak to 592 nm and increased absorbance at near-UV) and greater specific activity up to ~2X rates of wild type enzyme. The functional role of the loop is suggested to be in regulating oxidase activity. Considering the extensive similarity between AMMCO and NiR, particularly at the active site, engineering a trinuclear cluster into NiR appears feasible with a modest number of alterations to the polypeptide chain. With the aid of my newly developed highthroughput screening technique and site-directed mutagenesis, the mononuclear NiR active site was remodelled into a trinuclear Cu site similar to that of MCO. The substitutions Asp98His, Ile257His, Val304His, Asn305Thr and a 104-109 loop truncation were sufficient to allow for trinuclear cluster formation. A crystal structure of this variant was solved to 2.0 Å and the presence of three copper atoms at the engineered cluster was confirmed by Cu-edge anomalous diffraction data. Although the trinuclear copper cluster is present and catalyzes the reduction of oxygen, achieving rates of catalysis seen in iv native MCOs has proven more difficult. With the framework provided, further engineering NiR into a robust MCO is likely to provide further insights into the structural basis of oxygen reduction by trinuclear copper sites.