The crystal structures of cytochrome c oxidase from both bovine and Paracoccus denitrificans reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase. In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome c oxidase from Rhodobacter sphaeroides. A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a 3 (Fe 4؉ ‫؍‬ O 2؊ ) to the oxidized form (Fe 3؉ OH ؊ ). This redox step requires the delivery of a ''chemical'' H ؉ to protonate the reduced oxygen atom and is also coupled to proton pumping. It is found that mutations in the K channel (K362M and T359A) have virtually no effect on the ferryl-oxo-to-oxidized (F-to-Ox) transition, although steady-state turnover is severely limited. In contrast, electrogenic proton transfer at this step is strongly

The reaction with dioxygen of solubilized fully-reduced wild-type and EQ(I-286) (exchange of glutamate 286 of subunit I for glutamine) mutant cytochrome c oxidase from Rhodobacter sphaeroides has been studied using the flow-flash technique in combination with optical absorption spectroscopy. Proton uptake was measured using a pH-indicator dye. In addition, internal electron-transfer reactions were studied in the absence of oxygen. Glutamate 286 is found in a proton pathway proposed to be used for pumped protons from the crystal structure of cytochrome c oxidase from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669; E278 in P.d. numbering]. It is the residue closest to the oxygen-binding binuclear center that is clearly a part of the pathway. The results show that the wild-type enzyme becomes fully oxidized in a few milliseconds at pH 7.4 and displays a biphasic proton uptake from the medium. In the EQ(I-286) mutant enzyme, electron transfer after formation of the peroxy intermediate is impaired, CuA remains reduced, and no protons are taken up from the medium. Thus, the results suggest that E(I-286) is necessary for proton uptake after formation of the peroxy intermediate and transfer of the fourth electron to the binuclear center. The results also indicate that the proton uptake associated with formation of the ferryl intermediate controls the electron transfer from CuA to heme a.

SummaryPlant hormones interact at many different levels to form a network of signaling pathways connected by antagonistic and synergistic interactions. Ethylene and jasmonic acid both act to regulate the plant's responsiveness to a common set of biotic stimuli. In addition ethylene has been shown to negatively regulate the plant's response to the rhizobial bacterial signal, Nod factor. This regulation occurs at an early step in the Nod factor signal transduction pathway, at or above Nod factor-induced calcium spiking. Here we show that jasmonic acid also inhibits the plant's responses to rhizobial bacteria, with direct effects on Nod factor-induced calcium spiking. However, unlike ethylene, jasmonic acid not only inhibits spiking but also suppresses the frequency of calcium oscillations when applied at lower concentrations. This effect of jasmonic acid is amplified in the ethylene-insensitive mutant skl, indicating an antagonistic interaction between these two hormones for regulation of Nod factor signaling. The rapidity of the effects of ethylene and jasmonic acid on Nod factor signaling suggests direct crosstalk between these three signal transduction pathways. This work provides a model by which crosstalk between signaling pathways can rapidly integrate environmental, developmental and biotic stimuli to coordinate diverse plant responses.