Fig. 5 Two equivalents of PcuC·Cu1+·Cu2+ are required for conversion of ScoI·Cu2+·CoxB to CoxB·CuA. Two equivalents of PcuC·Cu1+·Cu2+ are required for.

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Fig. 5 Two equivalents of PcuC·Cu1+·Cu2+ are required for conversion of ScoI·Cu2+·CoxB to CoxB·CuA. Two equivalents of PcuC·Cu1+·Cu2+ are required for conversion of ScoI·Cu2+·CoxB to CoxB·CuA. (A) Mechanism of in vitro formation of CoxB·CuA (97% yield), deduced from the experiments shown in (B to H). (B and F) Titration of ScoI·Cu2+·CoxB (20 μM) with PcuC·Cu1+·Cu2+ at pH 7.0 and 25°C, analyzed by gel filtration (protein detection at 280 nm). ScoI·Cu2+·CoxB dissociation was completed after addition of one PcuC·Cu1+·Cu2+ equivalent. (C) Gel filtration runs from (B) but with protein detection at the CoxB·CuA–specific absorbance maximum at 813 nm. CoxB·CuA formation only reached its maximum after addition of two PcuC·Cu1+·Cu2+ equivalents to ScoI·Cu2+·CoxB. (D) Absorption spectra of the isolated CoxB·CuA peaks from (B) and (C), showing that two equivalents of PcuC·Cu1+·Cu2+ added to ScoI·Cu2+·CoxB quantitatively reconstituted the CoxB·CuA center (absorbance maxima at 367, 479, and 813 nm). (E) CoxB·CuA peak areas from (C) plotted against the added equivalents of PcuC·Cu1+·Cu2+. (G) Stopped-flow absorbance kinetics of the dissociation of the ScoI·Cu2+·CoxB complex (25 μM) by 1 equivalent (25 μM) of PcuC·Cu1+·Cu2+, yielding a second-order rate constant of 6.83 ± 0.02 × 102 M−1 s−1 (see Materials and Methods for the details). (H) Detection of cytochrome oxidase activity in B. diazoefficiens wild type (WT; positive control) and deletion mutants deficient in ScoI or PcuC. A coxB deletion mutant served as a negative control. Bacteria were grown aerobically in peptone–salt–yeast extract medium with or without 50 μM CuCl2. Identical amounts of cells were spotted onto a filter paper soaked with the indicator dye TMPD that reacts to indophenol blue when CoxB is active. Fabia Canonica et al. Sci Adv 2019;5:eaaw8478 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).