Structural and nitrite reductase activity comparisons of myoglobins with one to three distal histidines

Abstract

Although with a distinct heme active site, both myoglobin (Mb) and cytochrome c oxidase (CcO) were found to function as a nitrite reductase (NIR) under hypoxic conditions. On the other hand, Mb was rationally designed to mimic native CcO by introduction of two distal histidines, i.e., L29H/F43H mutant, where His29, His43 and native His64 formed a metal-binding site. To probe the role of distal histidines in regulating the NIR activity of Mb, we herein designed a single mutant of L29H Mb that contains two distal histidines and solved its X-ray crystal structure, then we made both structural and NIR activity comparisons of Mbs with one to three distal histidines. It was shown that introduction of one or two additional distal histidines in Mb leads to the formation of a distal hydrogen network, which slightly alters the conformation of heme active site and inhibits the NIR activity. Meanwhile, the reductase activity of both L29H Mb and L29H/F43H Mb are regulated by metal ions such as Cu(II) or Zn(II) binding to the distal histidines, i.e., Cu(II) enhances the reactivity while Zn(II) inhibits the reactivity. These findings provide valuable insights into the structure and function relationship for both Mb and CcO functioning as a NIR in biological system.