Reconstitution of the type-1 active site of the H145G/A variants of nitrite reductase by ligand insertion.
Variants of the copper-containing nitrite reductase (NiR) of Alcaligenes faecalis S6 were constructed by site-directed mutagenesis, by which the C-terminal histidine ligand (His145) of the Cu in the type-1 site was replaced by an alanine or a glycine. The type-1 sites in the NiR variants as isolated, are in the reduced form, but can be oxidized in the presence of external ligands, like (substituted) imidazoles and chloride. The reduction potential of the type-1 site of NiR-H145A reconstituted with imidazole amounts to 505 mV vs NHE (20 degrees C, pH 7, 10 mM imidazole), while for the native type-1 site it amounts to 260 mV. XRD data on crystals of the reduced and oxidized NiR-H145A variant show that in the reduced type-1 site the metal is 3-coordinated, but in the oxidized form takes up a ligand from the solution. With the fourth (exogenous) ligand in place the type-1 site is able to accept electrons at about the same rate as the wt NiR, but it is unable to pass the electron onto the type-2 site, leading to loss of enzymatic activity. It is argued that the uptake of an electron by the mutated type-1 site is accompanied by a loss of the exogenous ligand and a concomitant rise of the redox potential. This rise effectively traps the electron in the type-1 site.
Wijma
Hein J
HJ
Gorlaeus Laboratories, Metallo Protein Group, Leiden University, P. O. Box 9502, 2300 RA Leiden, The Netherlands.
Boulanger
Martin J
MJ
Molon
Annamaria
A
Fittipaldi
Maria
M
Huber
Martina
M
Murphy
Michael E P
ME
Verbeet
Martin Ph
MP
Canters
Gerard W
GW
D000421:Alcaligenes / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D003461:Crystallography; D017931:DNA Primers; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009572:Nitrite Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation
D017931:DNA Primers; D008024:Ligands; D009572:Nitrite Reductases