Significant increase of oxidase activity through the genetic incorporation of a tyrosine-histidine cross-link in a myoglobin model of heme-copper oxidase

Abstract

Heme-copper oxidase (HCO) performs efficient four-electron reduction of oxygen to water without releasing toxic, reactive oxygen species (ROS). Essential for this function is a post-translationally modified histidine–tyrosine cross-link (Tyr-His) in its heme a3/Cu_B oxygen reduction center. Through the genetic incorporation of the Tyr-His ligand and Cu_B site into myoglobin, we recapitulated important features of HCO into this small soluble protein, which exhibits selective O₂ reduction activity while generating less than 6% ROS, at more than 1000 turnovers. These results support that Tyr-His crosslink is indeed important for HCO function, and creates the exciting opportunity to rapidly evolve better HCO model proteins to achieve higher activity and selectivity, which may be suitable as alternatives to precious metal catalyst in fuel cells.

Figure 1

(A) Cu_B site of cytochrome c oxidase (bovine numbering). (B) Structure model overlay of imiTyrCu_BMb (cyan) and F33YCu_BMb (yellow). (C) Synthesis of imiTyr 1. a) Di-tert-butyl dicarbonate, THF, 1N NaOH rt, 4 h. b) imidazole, Cs₂CO₃, CuI, DMF, reflux, 16 h.

Figure 1

(A) Cu_B site of cytochrome c oxidase (bovine numbering). (B) Structure model overlay of imiTyrCu_BMb (cyan) and F33YCu_BMb (yellow). (C) Synthesis of imiTyr 1. a) Di-tert-butyl dicarbonate, THF, 1N NaOH rt, 4 h. b) imidazole, Cs₂CO₃, CuI, DMF, reflux, 16 h.

Figure 2

(A) Coomassie-stained SDS-PAGE of TAG4 mutant (left) and mutant (right) myoglobin (indicated by black arrow) expression in the presence and absence of 1 mM UAA 1. (B) ESI-MS spectra of the TAG4 mutant. The insert shows the deconvoluted spectrum; expected mass: 18496 Da, found: 18497 Da.

Figure 3

(A) Rates of oxygen reduction to form either water (blue) or ROS (red) catalyzed by 6 μM wtMb, F33YCu_BMb, or imiTyrCu_BMb, in the presence or absence of 6 μM of Cu²⁺, with 0.6 mM TMPD and 6 mM ascorbic acid as reductants (B) O₂ reduction turnover number catalyzed by imiTyrCu_BMb or F33YCu_BMb.

Figure 3

(A) Rates of oxygen reduction to form either water (blue) or ROS (red) catalyzed by 6 μM wtMb, F33YCu_BMb, or imiTyrCu_BMb, in the presence or absence of 6 μM of Cu²⁺, with 0.6 mM TMPD and 6 mM ascorbic acid as reductants (B) O₂ reduction turnover number catalyzed by imiTyrCu_BMb or F33YCu_BMb.