Effects of site-directed mutations on heme reduction in Escherichia coli nitrate reductase a by menaquinol:: A stopped-flow study

We have studied the effects of site-directed mutations in Escherichia coli nitrate reductase A (NarGHI) on heme reduction by a menaquinol analogue (menadiol) using the stopped-flow method. For NarGHI(H66Y) and NarGHI(H187Y), both lacking heme b(L) but having heme b(H), the heme reduction by menadiol is abolished. For NarGHI(H56R) and NarGHI(H205Y), both without heme b(H) but with heme b(L) a smaller and slower heme reduction compared to that of the wild-type enzyme is observed. These results indicate that electrons from menadiol oxidation are transferred initially to heme b(L). A transient species, likely to be associated with a semiquinone radical anion, was generated not only on reduction of the wild-type enzyme as observed previously (1) but also on reduction of NarGHI(H56R) and NarGHI(H205Y). The inhibitors 2-n-heptyl-4-hydroxyquinoline-N-oxide and stigmatellin both have significant effects on the reduction kinetics of NarGHI(H56R) and NarGHI(H205Y). We have also investigated the reoxidation of menadiol-reduced heme by nitrate in the mutants. Compared to the wild type, no significant heme reoxidation is observed for NarGHI(H56R) and NarGFI(H205Y). This result indicates that a single mutation removing heme b(H) blocks the electron-transfer pathway from the subunit NarI to the catalytic dimer NarGH.