The b-cytochromes of plant mitochondria. A spectrophotometric and potentiometric study.

Abstract The b-cytochromes of gradient-purified plant mitochondria have been studied with the aim of resolving the different components and of clarifying some aspects of their response to antimycin and to changes in mitochondrial energy state. It is shown by appropriate difference spectra that mung bean and potato mitochondria contain four b-components associated with the respiratory chain (α-peaks, 25°, at 556, 558, 560, and 566 nm) and one or more b-components reduced only by dithionite (α-peak, 25°, at 557 to 561 nm). Experiments examining reduction of the b-cytochromes by different substrates demonstrate that b-556 and b-560 are high potential species compared to the other b-components and also lead to the conclusion that plant mitochondria contain an "accessibility barrier" preventing rapid oxidation-reduction equilibrium between the b-cytochromes and pyridine nucleotides. Further characterization of the plant b-cytochromes included potentiometric titrations on potato mitochondria at wavelength pairs corresponding to the major b-species (556–540, 560–540, and 566–575 nm). These titrations gave the following apparent midpoint potentials: b-556, 88 ± 3 mv; b-560, 79 ± 1 mv; b-566, -76 ± 6 mv. b-558 could not be resolved potentiometrically from the dithionite-reducible cytochrome b, and both components appeared to have midpoint potentials of about -100 mv. In connection with these experiments, the validity of assigning midpoint potentials from the titrations directly to individual cytochromes is discussed and control experiments relevant to such assignments are presented. Antimycin is found to affect the plant b-cytochromes in two ways: (a) it causes a 1- to 2-nm red shift in the α-peak of reduced cytochrome b-560, and (b) in succinate-reduced mitochondria, acting in combination with an oxidizing agent, it causes enhanced reduction of b-558 and b-566. In contrast to previous results (Bonner, W.D., Jr., and Slater, E.C. (1970) Biochim. Biophys. Acta 223, 349–353) for potatoes, the enhanced reduction of b-566 is found to have a sigmoidal concentration dependence in both uncoupled mitochondria and in coupled mitochondria supplemented with ATP. The response of the plant b-558 and b-566 components to changes in energy state is of particular interest because of recent proposals, based on results with animal mitochondria, that these components play a direct role in energy transduction (Chance, B., Wilson, D.F., Dutton, P.L., and Erecinnska, M. (1970) Proc. Nat. Acad. Sci. U.S.A. 66, 1175–1182). In anaerobic, succinate-reduced plant mitochondria, reduction of b-558 and b-566 is found to be increased when the respiratory chain is energized. In mung bean mitochondria, this extra reduction could be driven either by exogenous ATP or by coupled respiration, but in potato mitochondria the respiratory chain appeared to be inaccessible to exogenous ATP and could be energized only by coupled respiration. Under appropriate experimental conditions, the ATP-induced reduction in mung bean mitochondria was shown to be rapidly relaxed by addition of an oxidation-reduction mediator (phenazine methosulfate), suggesting that the reduction is merely due to reversed electron transport. Thus, the situation is markedly different from that in animal mitochondria where a similar enhanced reduction of b-558 and b-566 has been correlated with a large increase in their apparent midpoint potentials and where the latter effect is the primary evidence that these components function in energy transduction. The present work, in conjunction with the report of Dutton and Storey (Dutton, P.L., and Storey, B.T. (1971) Plant Physiol. 47, 282–288) that the apparent midpoint potential of mung bean b-566 does not increase in response to ATP, indicates that there is no evidence that b-558 and b-566 play such a role in plant mitochondria.