Impact of residues remote from the catalytic centre on enzyme catalysis of copper nitrite reductase

Abstract

Enzyme mechanisms are often probed by structure-informed point mutations and measurement of their effects on enzymatic properties to test mechanistic hypotheses. In many cases, the challenge is to report on complex, often inter-linked elements of catalysis. Evidence for long-range effects on enzyme mechanism resulting from mutations remains sparse, limiting the design/redesign of synthetic catalysts in a predictable way. Here we show that improving the accessibility of the active site pocket of copper nitrite reductase by mutation of a surface-exposed phenylalanine residue (Phe306), located 12 Å away from the catalytic site type-2 Cu (T2Cu), profoundly affects intra-molecular electron transfer, substrate-binding and catalytic activity. Structures and kinetic studies provide an explanation for the lower affinity for the substrate and the alteration of the rate-limiting step in the reaction. Our results demonstrate that distant residues remote from the active site can have marked effects on enzyme catalysis, by driving mechanistic change through relatively minor structural perturbations.

Recent structural insights into the function of copper nitrite reductases

“…residue some 12 Å away from T2Cu in AxNiR resulted in a change to the second coordination sphere of the Cu with consequent effects on the rate-limiting step of the enzyme reaction. 59 In this F306C mutant, the substrate access channel is more open and contains an additional water bound to the T2Cu-coordinating water molecule. The specific activity was remarkably increased four-fold while the apparent nitrite affinity and the rate of intramolecular ET were lower than in native AxNiR.…”

Causes of evolutionary rate variation among protein sites

“…For example, McLaughlin, Jr., et al 95 have shown that functionally important residues are generally less mutationally tolerant than are residues with less stringent functional constraint. Leferink et al 141 demonstrated that mutations which increase solvent accessibility at an active site have strong influences on an enzyme's catalytic efficiency, demonstrating a tight relationship between evolutionary rate and function.…”

Identification of a tyrosine switch in copper-haem nitrite reductases

“…This channel, which is approximately 6 Å wide, is hydrophobic and is formed by residues from two adjacent monomers. The architecture of this channel has been shown to be important in controlling the coordination geometry of bound nitrite as 2 -O,O or end-on 1 -O (Antonyuk et al, 2005;Fukuda et al, 2014;Boulanger & Murphy, 2003) and in determining the rate-limiting step in turnover (Leferink et al, 2014).…”