Fig. 3.

Difference spectroscopy of the reaction intermediates of CcO. (A) Absorption difference spectra in the visible wavelength range. The two-electron reduced CO-bound state (R₂CO) was generated by bubbling CO through a detergent solubilized solution of CcO. The visible spectrum (red trace) exhibits characteristic maxima at 590 nm of the α-band and at 432 nm in the Soret region. The shoulder on the latter band at 448 nm indicates some overreduction after the time-resolved IR experiments. P_m was created by applying a saturated solution of equimolar amounts of CO and O₂ to a protein–lipid film adhered to the inner surface of a quartz cuvette. The corresponding difference spectrum (P_m-O, middle trace) shows positive bands at 437 and 607 nm. F was established by the action of H₂O₂ on the protein-lipid film. The F-O difference spectrum (black trace, bottom) shows characteristic maxima at 434 and 580 nm. (B) Infrared spectra of CcO from R. sphaeroides, showing the E-R₂CO (red trace, top), P_m-O (blue trace, middle), and F-O (black trace, bottom) differences. (C) Enlarged view (×10) of the difference bands of wild-type CcO in the region where carbonyl vibrations of protonated carboxylic acids are expected. The dashed spectra are the corresponding spectra of the E286D mutant measured under identical conditions. The dashed vertical line indicates the frequency of the stretching vibration of the carboxylic acid side chain of E286. For comparison, the change in the carbonyl stretch of E286 in the transition from the oxidized to the fully reduced state (R₄-O) is shown in green [top traces; the fully reduced state has been prepared as published (20) but at pH 8.5].