Site-directed mutagenesis of histidine residues involved in Cu(II) binding and reduction by sperm whale myoglobin.
Sperm whale myoglobin (Mb) reduces Cu(II) through a site-specific mechanism involving complexation by one or more surface histidine residues. Three mutants of Mb, derived from recombinant wild-type Mb, were designed in which surface histidine residues exhibiting strong Cu(II) binding were replaced with amino acids with comparatively poor metal binding characteristics. The kinetics of Cu(II)(Gly)2 reduction by native Mb, recombinant wild-type Mb, and the mutants were compared. Recombinant wild-type Mb reduced Cu(II) at a rate similar to that of native Mb. Two single mutations (His-48----Ala and His-116----Asp) decreased the rate by 31% and 7%, respectively, relative to wild-type Mb and decreased the rate by 38% and 16%, respectively, relative to native Mb. A double mutation (His-113----Ala, His-116----Asp) decreased the rate only slightly more than the single mutation at His-116. Previous NMR studies showed that His-113 exhibits the strongest Cu(II) binding of all surface histidines, but the present experiments suggest that it plays little or no role in the reduction of Cu(II) by Mb. His-48, located 12.7 A from the Fe(II)-heme, participates in one-third of the redox activity of the protein. His-116 appears to play a minor role in the overall redox activity of Mb, but its involvement shows that Mb has the ability to reduce Cu(II) through a histidine residue located more than 20 A from the Fe(II)-heme. These experiments demonstrate that electron transport from the Fe(II)-heme to site-specifically bound Cu(II) can be mediated through multiple pathways in sperm whale Mb.
Van Dyke
B R
BR
Department of Biology, University of California, San Diego, La Jolla 92093.
Bakan
D A
DA
Glover
K A
KA
Hegenauer
J C
JC
Saltman
P
P
Springer
B A
BA
Sligar
S G
SG
D000818:Animals; D002413:Cations, Divalent; D003300:Copper / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry*; D066298:In Vitro Techniques; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D014907:Whales
D002413:Cations, Divalent; D009211:Myoglobin; D011994:Recombinant Proteins; D006639:Histidine; D003300:Copper