FIGURE 4. Kinetics of heme b_H following flash excitation of chromatophores from wild type and selected Asn-221 mutant strains.

Chromatophores were suspended in 4 ml of a medium containing 100 mM KCl, 50 mM MOPS buffer, pH 7.0, in an anaerobic cuvette poised at E_h of 120 ± 10 mV, with mediators as follows: 1,4-p-benzoquinone, 1,2-naphthoquinone 4-sulfonate, 1,2-naphthoquinone, and 1,4-naphthoquinone at 10 μM; N,N,N′,N′-tetramethyl-p-phenylenediamine, N-methyl phenazonium methosulfate, N-ethyl phenazonium methosulfate, pyocyanine, and 2,3,5,6-tetremethyl-p-phenylenediamine at 1 μM; Fe-EDTA at 100 μM. The redox poise was controlled by small additions of dithionite or ferricyanide. Nigericin and valinomycin were included at 10 μM to minimize electrochromic changes. The kinetics of reduction and oxidation of heme b_H were measured from absorbance changes following flash excitation, as the difference kinetics at 561–569 nm. Concentrations of heme b_H were calculated using an extinction coefficient of 20 mM⁻¹ cm⁻¹. Rates were normalized to [bc₁ complex] using the amplitude of heme b_H reduction in the presence of antimycin at 50 ms. Where indicated, antimycin A was added at 10 μM (red traces). The traces in the absence of inhibitor (black) represent the convolution of reduction and oxidation phases. The re-oxidation (blue traces) could be visualized by subtraction of the black trace from the red trace for each pair. Traces shown are an average from four measurements. Times indicated are with reference to a 5-μs xenon flash (cut-off filter to allow transmission of λ > 860 nm), given at zero time. A complementary filter screened the photodiode detector from the flash.