T2Cu Site Research Articles

Crystallography with Online Optical and X-ray Absorption Spectroscopies Demonstrates an Ordered Mechanism in Copper Nitrite Reductase

Nitrite reductases are key enzymes that perform the first committed step in the denitrification process and reduce nitrite to nitric oxide. In copper nitrite reductases, an electron is delivered from the type 1 copper (T1Cu) centre to the type 2 copper (T2Cu) centre where catalysis occurs. Despite significant structural and mechanistic studies, it remains controversial whether the substrates, nitrite, electron and proton are utilised in an ordered or random manner. We have used crystallography, together with online X-ray absorption spectroscopy and optical spectroscopy, to show that X-rays rapidly and selectively photoreduce the T1Cu centre, but that the T2Cu centre does not photoreduce directly over a typical crystallographic data collection time. Furthermore, internal electron transfer between the T1Cu and T2Cu centres does not occur, and the T2Cu centre remains oxidised. These data unambiguously demonstrate an ‘ordered’ mechanism in which electron transfer is gated by binding of nitrite to the T2Cu. Furthermore, the use of online multiple spectroscopic techniques shows their value in assessing radiation-induced redox changes at different metal sites and demonstrates the importance of ensuring the correct status of redox centres in a crystal structure determination. Here, optical spectroscopy has shown a very high sensitivity for detecting the change in T1Cu redox state, while X-ray absorption spectroscopy has reported on the redox status of the T2Cu site, as this centre has no detectable optical absorption.

Shall We Dance? How A Multicopper Oxidase Chooses Its Electron Transfer Partner

Multicopper oxidases (MCOs) are encoded in the genomes of Eukarya, Bacteria, and Archea. These proteins are unique in that they contain at least four Cu atom prosthetic groups organized into one each of the three spectral classifications of copper sites in biology: type 1 (T1), type 2 (T2), and binuclear type 3 (T3), where the T2 and T3 sites form a trinuclear Cu cluster. With these four redox-active copper sites, the multicopper oxidases catalyze the four-electron (4e(-)) reduction of dioxygen to 2H2O, an activity that they alone share with the terminal heme-containing oxidases. Most MCOs exhibit broad specificity towards organic reductants, while a relatively small number of family members exhibit equally robust activity towards metal ions like Fe(II), Cu(I), and Mn(II) and, thus, are considered metallo-oxidases. This Account analyzes the structure-activity features of multicopper oxidases that determine their relative substrate specificity. Since the substrate oxidation step involves an outer-sphere electron transfer from the reductant to the T1Cu site in the protein, the concepts of Marcus theory are applied to unravel the origin of the substrate specificity of the multicopper ferroxidases.

PH-profile crystal structure studies of C-terminal despentapeptide nitrite reductase from Achromobacter cycloclastes

Crystal structures of C-terminal despentapeptide nitrite reductase (NiRc-5) from Achromobacter cycloclastes were determined from 1.9 to 2.3 Å at pH 5.0, 5.4, and 6.2. NiRc-5, that has lost about 30% activity, is found to possess quite similar trimeric structures as the native enzyme. Electron density and copper content measurements indicate that the activity loss is not caused by the release of type 2 copper (T2Cu). pH-profile structural comparisons with native enzyme reveal that the T2Cu active center in NiRc-5 is perturbed, accounting for the partial loss of enzyme activity. This perturbation likely results from the less constrained conformations of two catalytic residues, Asp98 and His255. Hydrogen bonding analysis shows that the deletion of five residues causes a loss of more than half the intersubunit hydrogen bonds mediated by C-terminal tail. This study shows that the C-terminal tail plays an important role in controlling the conformations around the T2Cu site at the subunit interface, and helps keep the optimum microenvironment of active center for the full enzyme activity of AcNiR.