Deciphering Interheme Electron-Transfer Pathways in a Diheme Peroxidase from Methane-Oxidizing Bacteria.

Bis-FeIV-forming enzymes represent an emerging paradigm in high-valent heme iron chemistry, in which two hemes separated by 15-20 Å are both oxidized to the FeIV state. A tryptophan positioned between the two hemes in two out of the three known enzymes to form this state, MauG and MbnH, has been proposed as the dominant pathway for interheme electron transfer. The third enzyme, BthA, instead has a serine at this position. Here, we used site-directed mutagenesis of Trp113 in MbnH to test whether this tryptophan is uniquely required for interheme electron transfer. Computational pathway analysis identified the possibility of an alternative, noncovalent route mediated by Arg283 and a heme propionate with possible electronic coupling comparable to that of the native tryptophan pathway. Consistent with this prediction, only simultaneous disruption of both the tryptophan and propionate-mediated pathways abolished bis-FeIV formation entirely. Moreover, only mutants capable of forming bis-FeIV retained the ability to turnover substrate, directly establishing this high-valent state as the catalytic intermediate responsible for substrate modification. These results demonstrate that an intervening tryptophan is not required for fast interheme electron transfer, and instead reveal an additional, noncovalent pathway that may be a general feature of diheme peroxidases.