Catalytic reduction of dioxygen with modified Thermus thermophilus cytochrome c552

• Cytochrome c552 modified to include an open iron coordination site can reduce O2 to H2O.
• Addition of hydrogen bond donors in the active site enhances catalysis.
• Peroxidase-like features proximal to the active site do not enhance O2 reduction.

Efficient catalysis of the oxygen reduction reaction (ORR) is of central importance to function in fuel cells. Metalloproteins, such as laccase (Cu) or cytochrome c oxidase (Cu/Fe–heme) carry out the 4H+/4e− reduction quite efficiently, but using large, complex protein frameworks. Smaller heme proteins also can carry out ORR, but less efficiently. To gain greater insight into features that promote efficient ORR, we expressed, characterized, and investigated the electrochemical behavior of six new mutants of cytochrome c552 from Thermus thermophilus: V49S/M69A, V49T/M69A, L29D/V49S/M69A, P27A/P28A/L29D/V49S/M69A, and P27A/P28A/L29D/V49T/M69A. Mutation to V49 causes only minor shifts to FeIII/II reduction potentials (E°′), but introduction of Ser provides a hydrogen bond donor that slightly enhances oxygen reduction activity. Mutation of L29 to D induces small shifts in heme optical spectra, but not to E°′ (within experimental error). Replacement of P27 and P28 with A in both positions induces a − 50 mV shift in E°′, again with small changes to the optical spectra. Both the optical spectra and reduction potentials have signatures consistent with peroxidase enzymes. The V49S and V49T mutations have the largest impact of ORR catalysis, suggesting that increased electron density at the Fe site does not improve O2 reduction chemistry.

Cytochrome c552 from Thermus thermophilus modified to include proton relay groups in the distal heme site catalyzes reduction of O2 at neutral pH.Figure optionsDownload as PowerPoint slide