The effect of glutaraldehyde fixation on the primary photochemical processes in bacterial photosynthesis

In Chromatium D the half-time for laser-induced (20–30-nsec flash) cytochrome C553 oxidation in redox poised chromatophores (1 μsec) and cytochrome C555 oxidation in whole cells (2.5μsec) is not affected by glutaraldehyde fixation. The reduction half-times for both cytochromes, however, increase as different functions of the glutaraldehyde concentration during the whole cell fixation process. At a cell-fixing concentration of 0.8%, cytochrome C555 but not C553 is observed after a laser flash. Steady light-induced spectra using similar preparations suggest the possibility of four components observable in the 500–620-nm range. These are cytochrome C555, P600, a species peaking at 560 nm and a component displaying a light-induced blue shift in the 500–540-nm region which may be a carotenoid response. The wavelength expected for the α-peak (reduced-minus-oxidized) of cytochrome cc′ is 560 nm, but the lack of a corresponding Soret peak makes identification uncertain and raises the possibility that we are observing a totally new component. Comparison of the amount of cytochrome oxidized by steady illumination and by a laser flash shows that on the average there are three cytochrome C555 molecules per reaction center in both whole cells and chromatophores. If the glutaraldehyde acts directly on the reaction center cytochromes then it is clear that cytochrome reduction requires large amplitude motion, but that oxidation does not. However, glutaraldehyde fixation may simply block the path of reducing electrons before they reach reaction center bound cytochromes.