Spectroscopic and Electronic Structure Studies of the μ₄-Sulfide Bridged Tetranuclear Cu_ZCluster in N₂O Reductase:  Molecular Insight into the Catalytic Mechanism

Abstract

Spectroscopic methods combined with density functional calculations are used to develop a detailed bonding description of the mu(4)-sulfide bridged tetranuclear Cu(Z) cluster in N(2)O reductase. The ground state of Cu(Z) has the 1Cu(II)/3Cu(I) configuration. The single electron hole dominantly resides on one Cu atom (Cu(I)) and partially delocalizes onto a second Cu atom (Cu(II)) via a Cu(I)-S-Cu(II) sigma/sigma superexchange pathway which is manifested by a Cu(II) --> Cu(I) intervalence transfer transition in absorption. The observed excited-state spectral features of Cu(Z) are dominated by the S --> Cu(I) charge-transfer transitions and Cu(I) based d-d transitions. The intensity pattern of individual S --> Cu(I) charge-transfer transitions reflects different bonding interactions of the sulfur valence orbitals with the four Cu's in the Cu(Z) cluster, which are consistent with the individual Cu-S force constants obtained from a normal coordinate analysis of the Cu(Z) resonance Raman frequencies and profiles. The Cu(I) d orbital splitting pattern correlates with its distorted T-shaped ligand field geometry and accounts for the observed low g( parallel ) value of Cu(Z) in EPR. The dominantly localized electronic structure description of the Cu(Z) site results from interactions of Cu(II) with the two additional Cu's of the cluster (Cu(III)/Cu(IV)), where the Cu-Cu electrostatic interactions lead to hole localization with no metal-metal bonding. The substrate binding edge of Cu(Z) has a dominantly oxidized Cu(I) and a dominantly reduced Cu(IV). The electronic structure description of Cu(Z) provides a strategy to overcome the reaction barrier of N(2)O reduction at this Cu(I)/Cu(IV) edge by simultaneous two-electron transfer to N(2)O in a bridged binding mode. One electron can be donated directly from Cu(IV) and the other from Cu(II) through the Cu(II)-S-Cu(I) sigma/sigma superexchange pathway. A frontier orbital scheme provides molecular insight into the catalytic mechanism of N(2)O reduction by the Cu(Z) cluster.

The tetranuclear copper active site of nitrous oxide reductase: the CuZ center

“…Cu I charge-transfer transition [59,70]. In particular, the three main frequency lines in the resonance Raman spectra, at 366, 386 and 415 cm -1 , are associated with Cu-S vibrations and contribute to the broad charge-transfer absorption band at approximately 640 nm (approximately 15,650 cm -1 ).…”

A new CuZ active form in the catalytic reduction of N2O by nitrous oxide reductase from Pseudomonas nautica

“…Cu ligand to metal charge transfer (LMCT) transition, as for the 640-nm band in the resting CuZ, which is actually a multicomponent band with partially overlapping features at higher and lower energy [11,12]. Indeed, looking carefully at the difference spectra reported in the inset in Fig.…”

“…nautica N 2 OR, in the as-isolated state, contains a 1Cu II /3Cu I redox state with a total spin of , where the unpaired electron is delocalized between two or more copper atoms through the bridging sulfide ion [10][11][12]. This state, which has a typical electronic spectral band at 640 nm and a four-line splitting EPR signature [13,14], cannot be easily reduced or oxidized.…”

Enzymatic activity mastered by altering metal coordination spheres

“…. Addition of dithionite reduces the CuA center with the CuZ center maintaining its formal oxidation state [64][65][66][67][68]. This form of the enzyme corresponds to an inactive form.…”

“…In order to be active, the enzyme requires prolonged incubation in the presence of reduced methyl viologen, which fully reduces CuZ to four Cu 1? [66]. There are two possible ways for the N 2 O molecule to bind to CuZ.…”