High and Low Energy States of Cytochromes: III. IN REACTIONS WITH CATIONS

Abstract

Upon addition of low concentrations of calcium or manganese to pigeon heart mitochondria in a phosphate-free medium, a characteristic pattern of steady state changes ensues which leads to a high absorbance at 560 mµ (at 300° K) characteristic of cytochrome b. Other components of the respiratory chain are highly oxidized; electron transport from succinate and glutamate is inhibited, as is energy transfer into the pathway for diphosphopyridine nucleotide reduction. The spectroscopic changes and the inhibition of electron transfer represent State 6. Low concentrations of cations are required for half-maximal change in cytochrome b; about 15 µ m calcium gives half-maximal effect. The calcium concentration required is a function of pH; the response of cytochrome b of the largest magnitude is observed at pH 8. High concentrations of calcium (50 to 150 µ m ) decrease this effect. The inhibited state is readily reversed by phosphate, leading to reduction of all the components of the respiratory chain except cytochrome b, which proceeds to a less absorbing state. Under these conditions, the functions of electron transport, energy-linked pyridine nucleotide reduction, and response to adenosine diphosphate are largely restored. The inhibited state of electron transfer is readily reactivated by uncoupling agents, but energy-linked pyridine nucleotide reduction is not reactivated. Examination of cytochrome b spectra at low temperature (77° K) indicates the accumulation of a compound absorbing at 555 mµ in the presence of low concentrations of cations and its transformation to a compound absorbing at 565 mµ upon addition of phosphate. Cytochrome c responds most rapidly to cations; cytochrome b and reduced pyridine nucleotide respond less rapidly, but at low concentrations of cations. The alkalinization of the cristal membrane (29) is a slower reaction that requires higher cation concentrations. Both the reaction of cytochromes with cations and the alkalinization of the cristal membrane are considered as factors in causing the inhibited state of electron transfer, State 6.