CO sensitivity of cytochrome a3 in the leaves of a number of C3 and C4 plants was monitored by the nitrate reductase assay under differing CO to 02 ratios. All the C3 plants were relatively insensitive to CO and required a high CO to 02 ratio of 40 to promote significant nitrate reductase activity. However, when treated with 2 millimolar 2,4-dinitrophenol, these leaves readily responded to CO even at low CO to 02 ratios of 10 or less. On the other hand, the leaves of all C4 plants tested, belonging to the three subgroups, were highly sensitive to CO, even at CO to 02 ratios of 5 or less. In these leaves, the uncoupler was without any effect, probably because the mitochondria, either from me sophyll or bundle sheath cells or both, lacked tight respiratory control.Cyt c oxidase (EC 1.9.3.1), the multiunit enzyme complex consisting of Cyt a and Cyt a3 and associated CuA and CuB components, is the terminal oxidase in the mitochondrial electron transfer chain and contains site 3 of oxidative phosphorylation (16). CO is a specific inhibitor of the enzyme, as it forms a complex with reduced Cyt a3 (1, 3). In mammalian mitochondria, this complex is considered to be stable in the dark but can be photodissociated after exposure to a flash of light with absorption maxima at 430 and 590 nm (2). We had recently observed that in a number of plant species, CO can be replaced from the active site in complete darkness when excess 02 is supplied (10). Pollok et al. (12) oxidase by CO, which in turn was a reflection of the reduced state of Cyt a3 during steady-state respiration. This technique was earlier used to examine CO-sensitivity of Cyt c oxidase in the leaves of wheat (9), pearl millet (7), and sorghum (8).In these studies, the leaves were exposed to CO briefly for 1 min and then incubated under aerobic conditions for the determination of nitrite accumulation in vivo. Hence, the relative concentrations of CO and 02 during incubation were not known with certainty. We have now examined a large number of C3 and C4 plants, exposed to precisely defined CO to 02 ratios during the course of incubation. We now report that there are distinct differences between C3 and C4 plants with regard to CO-sensitivity of Cyt c oxidase. Thus, the leaves of all C4 plants tested are highly sensitive even at low CO to 02 ratios, whereas in C3 plants, considerably higher CO concentrations are required to inhibit Cyt c oxidase.Because the redox state of Cyt a3 during steady-state respiration probably depends on the tightness of coupling of oxidative phosphorylation at site 3, the results suggest possible differences in this regard between the leaves of C3 and C4 plants.
