Tryptophan Can Promote Oxygen Reduction to Water in a Biosynthetic Model of Heme Copper Oxidases

Heme-copper oxidases (HCOs) utilize tyrosine (Tyr) to donate one of the four electrons required for the reduction of O-2 to water in biological respiration, while tryptophan (Trp) is speculated to fulfill the same role in cyt bd oxidases. We previously engineered myoglobin into a biosynthetic model of HCOs and demonstrated the critical role that Tyr serves in the oxygen reduction reaction (ORR). To address the roles of Tyr and Trp in these oxidases, we herein report the preparation of the same biosynthetic model with the Tyr replaced by Trp and further demonstrate that Trp can also promote the ORR, albeit with lower activity. An Xray crystal structure of the Trp variant shows a hydrogen-bonding network involving two water molecules that are organized by Trp, similar to that in the Tyr variant, which is absent in the crystal structure with the native Phe residue. Additional electron paramagnetic resonance measurements are consistent with the formation of a Trp radical species upon reacting with H2O2. We attribute the lower activity of the Trp variant to Trp's higher reduction potential relative to Tyr. Together, these findings demonstrate, for the first time, that Trp can indeed promote the ORR and provides a structural basis for the observation of varying activities. The results support a redox role for the conserved Trp in bd oxidase while suggesting that HCOs use Tyr instead of Trp to achieve higher reactivity.

Automated summary

A biosynthetic model of oxidases was constructed by replacing tyrosine with tryptophan, and it was demonstrated that tryptophan can also promote the oxygen reduction reaction, although with lower activity. Structural analysis and experimental results indicate that the difference in activity is due to the higher reduction potential of tryptophan. This study provides the first evidence that tryptophan can promote the oxygen reduction reaction and offers a structural basis for the observation of varying activities.