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 Å from the Fe(II)-herne, participates in one-third of the redox activity of the protein. His-116 appears to play a minor role hi 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 Å from the Fe(II)-heme. These experiments demonstrate that electron transport from the Fe(II)-heme to site-specifically bound Cu(H) can be mediated through multiple pathways in sperm whale Mb.