Simultaneous Monitoring of Oxidative Phosphorylation Chromophores: Regulation of State 3 Crossover in Complex III

Understanding reducing equivalent flux control within the electron transport chain requires simultaneous monitoring of all respiratory components. As such, we developed a spectrophotometric system comprising a center‐mounted cuvette in an integrating sphere to minimize the effects of scattering in turbid mitochondrial suspensions. By acquiring spectra every 100 ms spanning 400 to 700 nm, this system permits collection of cytochrome absorbance data in porcine heart mitochondria (1 nmol cyt a /mL) with redundant measurements in the alpha and Soret regions. Spectra were analyzed using a linear least squares fitting routine with model optical spectra of the electron transfer components (flavin adenine dinucleotide and cyt b H , b L , c 1 , c , and a ). A redox crossover event was observed between cyt b (more oxidized) and cyt c (more reduced) during the state 4 to 3 transition. Furthermore, this crossover could be altered by changes in matrix [Ca 2+ ]. In Ca 2+ ‐depleted mitochondria, the overall respiratory rate was lower and the crossover was at the more conventional site between cyt c and a . Upon replenishment of matrix Ca 2+ , the crossover point shifted back to cyt b and c , concomitant with a two‐ to three‐fold increase in state 3 respiratory rate. These data demonstrate the practicality of monitoring the entire redox chain in intact mitochondria and provide novel evidence that Ca 2+ may regulate electron transfer.