Figure 3. Spectro-electrochemical titrations of the WT Ec-cyt bd and the EcE445Q and EcE445C mutants.

The redox state of heme d was monitored by the absorption difference (ΔA) as a function of solution potential (panels A, C and D). ΔA was measured by using the following linear combination of wavelengths (A_{629 nm} – 0.5*(A_{660 nm} + A_{608 nm})). Similar trends were observed with the wavelength combinations proposed in [29]. The redox state of heme b₅₅₈ was monitored by the absorption difference (ΔA) as a function of solution potential (panel B), where ΔA = A_{561 nm} – 1.6*A_{573 nm} + 0.06*A_{629 nm}). Again, all trends were verified by comparison to midpoint potentials calculated using other wavelength combinations mentioned in [29]. Data were fit to a 1-electron reduction following the Nernst equation and the results are summarized in Table 2.