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.

A rapid and highly efficient method of purifying the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been developed. This method relies upon a six-histidine affinity tag fused to the C-terminus of subunit I, which confers to the Ni -nltrdotriacetic acid (NTA) agaoxidase a high affinity for .2+ • • rose. The histidine-tagged oxidase can be purified rapidly and with high yield by one affinity chromatography step, starting with solubilized membranes. The purified oxidase is > 95% pure and possesses structural and functional characteristics of the wildtype enzyme. The six-histidine tag can be easily added to pre-constructed site-directed mutants of subunit I, increasing the availability of purified cytochrome c oxidase mutants for biophysical and biochemical studies.

Resonance Raman spectra of the carbon monoxy derivative of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides show two distinct Fe-CO stretching modes (519 and 493 cm-1) at room temperature. The frequency of the mode at 519 cm-1 coincides with that of other terminal oxidases at neutral pH. Two C-O stretching modes, one at 1966 cm-1 and one at 1955 cm-1, are also found. The splitting of the C-O stretching mode is consistent with the FTIR spectra of cytochrome c oxidases at cryogenic temperatures in which two different conformations (alpha and beta) of the catalytic site of the enzyme are present. The splitting of both the Fe-CO and C-O stretching modes under our conditions indicates that these two forms of the enzyme are also present at room temperature, and with the additional information on the Fe-CO modes provided here, a structural origin for the two forms may be postulated. The alpha-form has the same general structure of the active site as mammalian oxidase, a structure in which the copper atom that is the part of the Fe-CuB binuclear site interacts strongly with the bound CO. We postulate that the copper atom exerts a strong polar or steric effect on the heme-bound CO, resulting in either compression of the Fe-CO bond or distortion of the Fe-CO moiety.(ABSTRACT TRUNCATED AT 250 WORDS)

The aa3-type cytochrome c oxidase from Rhodobacter sphaeroides is closely related to eukaryotic cytochrome c oxidases. Analysis of site-directed mutants identified the ligands of heme a, heme a3, and CuB [Hosler et al. (1993) J. Bioenerg. Biomembr. 25, 121-133], which have been confirmed by high-resolution structures of homologous oxidases [Iwata et al. (1995) Nature 376, 660; Tsukihara et al. (1995) Science 269, 1069; (1996) 272, 1136]. Since the protons used to form water originate from the inner side of the membrane, and the heme a3-CuB center is located near the outer surface, the protein must convey these substrate protons to the oxygen reduction site. Transmembrane helix VIII in subunit I is close to this site and contains several conserved polar residues that could function in a rate-determining proton relay system. To test this role, apolar residues were substituted for T352, T359, and K362 in helix VIII and the mutants were characterized in terms of activity and structure. Mutation of T352, near CuB, strongly decreases enzyme activity and disrupts the spectral properties of the heme a3-CuB center. Mutation of T359, below heme a3, substantially reduces oxidase activity with only minor effects on metal center structure. Two mutations of K362, approximately 15 A below the axial ligand of heme a3, are inactive, make heme a3 difficult to reduce, and cause changes in the resonance Raman signal specific for the iron-histidine bond to heme a3. The results are consistent with a key role for T352, T359, and K362 in oxidase activity and with the involvement of T359 and K362 in proton transfer through a relay system now plausibly identified in the crystal structure. However, the characteristics of the K362 mutants raise some questions about the assignment of this as the substrate proton channel.

The LATD gene of the model legume, Medicago truncatula, is required for the normal function of three meristems, i.e. the primary root, lateral roots and nitrogen-fixing nodules. In latd mutants, primary root growth eventually arrests, resulting in a disorganized root tip lacking a presumptive meristem and root cap columella cells. Lateral root organs are more severely affected; latd lateral roots and nodules arrest immediately after emerging from the primary root, and reveal a lack of organization. Here we show that the plant hormone, abscisic acid (ABA), can rescue the latd root, but not nodule, meristem defects. Growth on ABA is sufficient to restore formation of small, cytoplasm-rich cells in the presumptive meristem region, rescue meristem organization and root growth and formation of root cap columella cells. In contrast, inhibition of ethylene synthesis or signaling fails to restore latd primary root growth. We find that latd mutants have normal levels of ABA, but exhibit reduced sensitivity to the hormone in two other ABA-dependent processes: seed germination and stomatal closure. Together, these observations demonstrate that the latd mutant is defective in the ABA response and indicate a role for LATD-dependent ABA signaling in M. truncatula root meristem function.

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