Spectroscopically Validated pH-dependent MSOX Movies Provide Detailed Mechanism of Copper Nitrite Reductases.

Copper nitrite reductases (CuNiRs) exhibit a strong pH dependence of their catalytic activity. Structural movies can be obtained by serially recording multiple structures (frames) from the same spot of a crystal using the MSOX serial crystallography approach. This method has been combined with on-line single crystal optical spectroscopy to capture the pH-dependent structural changes that accompany during turnover of CuNiRs from two Rhizobia species.

A 2.2 Å cryoEM structure of a quinol-dependent NO Reductase shows close similarity to respiratory oxidases.

Quinol-dependent nitric oxide reductases (qNORs) are considered members of the respiratory heme-copper oxidase superfamily, are unique to bacteria, and are commonly found in pathogenic bacteria where they play a role in combating the host immune response. qNORs are also essential enzymes in the denitrification pathway, catalysing the reduction of nitric oxide to nitrous oxide. Here, we determine a 2.

Single crystal spectroscopy and multiple structures from one crystal (MSOX) define catalysis in copper nitrite reductases.

Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme.

Nature of the copper-nitrosyl intermediates of copper nitrite reductases during catalysis.

The design and synthesis of copper complexes that can reduce nitrite to NO has attracted considerable interest. They have been guided by the structural information on the catalytic Cu centre of the widespread enzymes Cu nitrite reductases but the chemically novel side-on binding of NO observed in all crystallographic studies of these enzymes has been questioned in terms of its functional relevance. We show conversion of NO to NO in the crystal maintained at 170 K and present 'molecular movies' defining events during enzyme turnover including the formation of side-on Cu-NO intermediate.

An unprecedented insight into the catalytic mechanism of copper nitrite reductase from atomic-resolution and damage-free structures.

Copper-containing nitrite reductases (CuNiRs), encoded by gene, are found in all kingdoms of life with only 5% of CuNiR denitrifiers having two or more copies of Recently, we have identified two copies of genes in several α-proteobacteria of the order Rhizobiales including sp. ORS 375, encoding a four-domain heme-CuNiR and the usual two-domain CuNiR ( NiR). Compared with two of the best-studied two-domain CuNiRs represented by the blue (NiR) and green (NiR) subclasses, NiR, a blue CuNiR, shows a substantially lower catalytic efficiency despite a sequence identity of ~70%.