pH Dependence of Copper Geometry, Reduction Potential, and Nitrite Affinity in Nitrite Reductase

Abstract

Many properties of copper-containing nitrite reductase are pH-dependent, such as gene expression, enzyme activity, and substrate affinity. Here we use x-ray diffraction to investigate the structural basis for the pH dependence of activity and nitrite affinity by examining the type 2 copper site and its immediate surroundings in nitrite reductase from Rhodobacter sphaeroides 2.4.3. At active pH the geometry of the substrate-free oxidized type 2 copper site shows a near perfect tetrahedral geometry as defined by the positions of its ligands. At higher pH values the most favorable copper site geometry is altered toward a more distorted tetrahedral geometry whereby the solvent ligand adopts a position opposite to that of the His-131 ligand. This pH-dependent variation in type 2 copper site geometry is discussed in light of recent computational results. When co-crystallized with substrate, nitrite is seen to bind in a bidentate fashion with its two oxygen atoms ligating the type 2 copper, overlapping with the positions occupied by the solvent ligand in the high and low pH structures. Fourier transformation infrared spectroscopy is used to assign the pH dependence of the binding of nitrite to the active site, and EPR spectroscopy is used to characterize the pH dependence of the reduction potential of the type 2 copper site. Taken together, these spectroscopic and structural observations help to explain the pH dependence of nitrite reductase, highlighting the subtle relationship between copper site geometry, nitrite affinity, and enzyme activity.

NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions

“…2E). The 115-mV increase in redox potential is in accordance with previous studies, showing that complexation of Cu 2+ in a type 2 copper binding site increases the redox potential by 40-240 mV relative to the potential of aqueous Cu 2+ (19)(20)(21). The fact that CBP21 seems to preferably bind Cu 1+ coincides with the notion that molecular oxygen tends to bind copper proteins in their reduced monovalent state (22).…”

The enzyme mechanism of nitrite reductase studied at single-molecule level

“…They can easily amount to plus or minus 50 mV, in line with recent observations on the range of midpoint potentials observed by FRET detected cyclic voltammetry on small clusters of azurin (34,37). With a typical value for the reorganization energy, , of 700 mV and a driving force ⌬G Ͻ Ͻ , a 100-mV variation in midpoint potential will affect the rate of electron transfer from the type 1 to the type 2 site by a factor of Ϸ2, as calculated from Marcus' theory.…”

“…Structural and conformational variations in the first and second coordination sphere of a metal are known to cause variations in midpoint potential (34). They can easily amount to plus or minus 50 mV, in line with recent observations on the range of midpoint potentials observed by FRET detected cyclic voltammetry on small clusters of azurin (34,37).…”

“…There are two main contributions to the B factor: increased mobility and static disorder (see also ref. 34). † † Partial disorder of water molecules means that in the electron density map, weak electron density is observed at some points that may correspond to water molecules.…”

“…In all NiRs studied to date, the type 2 Cu is immobilized in the protein framework by three histidines. When this site is oxidized, its fourth coordination position is occupied by a water molecule or a hydroxide ion at different positions, depending on pH (34). When the site is reduced, this fourth ligand disorders (18,33).…”