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Carbonyl region of M-BR difference spectra at pH 7.4 of the double mutant E9Q/E74Q and the single-site mutants E194Q and E204Q. As in the wild-type experiments (Fig. 4), M-BR difference spectra have been extracted from time-resolved experiments at 400 μs after the flash. The bottom spectrum is a steady-state M-BR difference spectrum of the quadruple mutant E9Q/E74Q/E194Q/E204Q (E4Q) taken at −18°C, pH 7, under continuous illumination with yellow light and the dark state as reference.
