Mutation of a single residue in the ba 3 oxidase specifically impairs protonation of the pump site

The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative "pump site" was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase.

New insights into the regulation of NADPH oxidase dependent reactive oxygen species signaling during the plant immune response

Las NADPH oxidasas de plantas, denominadas “respiratory burst oxidase homologues” (RBOHs), producen especies reactivas del oxígeno (ROS) que median un amplio rango de funciones. En la célula vegetal, el ajuste preciso de la producción de ROS aporta la especificidad de señal para generar una respuesta apropiada ante las amenazas ambientales. RbohD y RbohF, dos de los diez genes Rboh de Arabidopsis, son pleiotrópicos y median diversos procesos fisiológicos en respuesta a patógenos. El control espacio-temporal de la expresión de los genes RbohD y RbohF podría ser un aspecto crítico para determinar la multiplicidad de funciones de estas oxidasas. Por ello, generamos líneas transgénicas de Arabidopsis con fusiones de los promoters de RbohD y RbohF a los genes delatores de la B-glucuronidasa y la luciferasa. Estas líneas fueron empleadas para revelar el patrón de expresión diferencial de RbohD y RbohF durante la respuesta inmune de Arabidopsis a la bacteria patógena Pseudomonas syringae pv. tomato DC3000, el hongo necrótrofo Plectosphaerella cucumerina y en respuesta a señales relacionadas con la respuesta inmune. Nuestros experimentos revelan un patrón de expresión diferencial de los promotores de RbohD y RbohF durante el desarrollo de la planta y en la respuesta inmune de Arabidopsis. Además hemos puesto de manifiesto que existe una correlación entre el nivel de actividad de los promotores de RbohD y RbohF con la acumulación de ROS y el nivel de muerte celular en respuesta a patógenos. La expression de RbohD y RbohF también es modulada de manera diferencial en respuesta a patrones moleculares asociados a patógenos (PAMPs) y por ácido abscísico (ABA). Cabe destacar que, mediante una estrategia de intercambio de promotores, hemos revelado que la región promotora de RbohD, es necesaria para dirigir la producción de ROS en respuesta a P. cucumerina. Adicionalmente, la activación del promotor de RbohD en respuesta al aislado de P. cucumerina no adaptado a Arabidopsis 2127, nos llevó a realizar ensayos de susceptibilidad con el doble mutante rbohD rbohF que han revelado un papel desconocido de estas oxidasas en resistencia no-huesped. La interacción entre la señalización dependiente de las RBOHs y otros componentes de la respuesta inmune de plantas podría explicar también las distintas funciones que median estas oxidasas en relación con la respuesta inmune. Entre la gran cantidad de señales coordinadas con la actividad de las RBOHs, existen evidencias genéticas y farmacológicas que indican que las proteínas G heterotriméricas están implicadas en algunas de las rutas de señalización mediadas por ROS derivadas de los RBOHs en respuesta a señales ambientales. Por ello hemos estudiado la relación entre estas RBOH-NADPH oxidasas y AGB1, la subunidad β de las proteínas G heterotriméricas en la respuesta inmune de Arabidopsis. Análisis de epistasis indican que las proteínas G heterotriméricas están implicadas en distintas rutas de señalización en defensa mediadas por las RBOHs. Nuestros resultados ilustran la relación compleja entre la señalización mediada por las RBOHs y las proteínas G heterotriméricas, que varía en función de la interacción planta-patógeno analizada. Además, hemos explorado la posible asociación entre AGB1 con RBOHD y RBOHF en eventos tempranos de la respuesta immune. Cabe señalar que experimentos de coímmunoprecipitación apuntan a una posible asociación entre AGB1 y la kinasa citoplasmática reguladora de RBOHD, BIK1. Esto indica un posible mecanismo de control de la función de esta NADPH oxidase por AGB1. En conjunto, estos datos aportan nuevas perspectivas sobre cómo, a través del control transcripcional o mediante la interacción con las proteínas G heterotriméricas, las NADPH oxidases de plantas median la producción de ROS y la señalización por ROS en la respuesta inmune. Nuestro trabajo ejemplifica cómo la regulación diferencial de dos miembros de una familia multigénica, les permite realizar distintas funciones fisiológicas especializadas usando un mismo mecanismo enzimático. ABSTRACT The plant NADPH oxidases, termed respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) which mediate a wide range of functions. Fine tuning this ROS production provides the signaling specificity to the plant cell to produce the appropriate response to environmental threats. RbohD and RbohF, two of the ten Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes in response to pathogens. One aspect that may prove critical to determine the multiplicity of functions of RbohD and RbohF is the spatio-temporal control of their gene expression. Thus, we generated Arabidopsis transgenic lines with RbohD- and RbohF-promoter fusions to the β-glucuronidase and the luciferase reporter genes. These transgenics were employed to reveal RbohD and RbohF promoter activity during Arabidopsis immune response to the pathogenic bacterium Pseudomonas syringae pv tomato DC3000, the necrotrophic fungus Plectosphaerella cucumerina and in response to immunity-related cues. Our experiments revealed a differential expression pattern of RbohD and RbohF throughout plant development and during Arabidopsis immune response. Moreover, we observed a correlation between the level of RbohD and RbohF promoter activity, the accumulation of ROS and the amount of cell death in response to pathogens. RbohD and RbohF gene expression was also differentially modulated by pathogen associated molecular patterns and abscisic acid. Interestingly, a promoter-swap strategy revealed the requirement for the promoter region of RbohD to drive the production of ROS in response to P. cucumerina. Additionally, since the RbohD promoter was activated during Arabidopsis interaction with a non-adapted P. cucumerina isolate 2127, we performed susceptibility tests to this fungal isolate that uncovered a new role of these oxidases on non-host resistance. The interplay between RBOH-dependent signaling with other components of the plant immune response might also explain the different immunity-related functions mediated by these oxidases. Among the plethora of signals coordinated with RBOH activity, pharmacological and genetic evidence indicates that heterotrimeric G proteins are involved in some of the signaling pathways mediated by RBOH–derived ROS in response to environmental cues. Therefore, we analysed the interplay between these RBOH-NADPH oxidases and AGB1, the Arabidopsis β-subunit of heterotrimeric G proteins during Arabidopsis immune response. We carried out epistasis studies that allowed us to test the implication of AGB1 in different RBOH-mediated defense signaling pathways. Our results illustrate the complex relationship between RBOH and heterotrimeric G proteins signaling, that varies depending on the type of plant-pathogen interaction. Furthermore, we tested the potential association between AGB1 with RBOHD and RBOHF during early immunity. Interestingly, our co-immunoprecipitation experiments point towards an association of AGB1 and the RBOHD regulatory kinase BIK1, thus providing a putative mechanism in the control of the NADPH oxidase function by AGB1. Taken all together, these studies provide further insights into the role that transcriptional control or the interaction with heterotrimeric G-proteins have on RBOH-NADPH oxidase-dependent ROS production and signaling in immunity. Our work exemplifies how, through a differential regulation, two members of a multigenic family achieve specialized physiological functions using a common enzymatic mechanism.

The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer

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 a3 (Fe4+ = O2−) to the oxidized form (Fe3+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 suppressed by mutations in the D channel. The results strongly suggest that the functional roles of the two channels are not the separate delivery of chemical or pumped protons, as proposed recently [Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. (1995) Nature (London) 376, 660–669]. The D channel is likely to be involved in the uptake of both “chemical” and “pumped” protons in the F-to-Ox transition, whereas the K channel is probably idle at this partial reaction and is likely to be used for loading the enzyme with protons at some earlier steps of the catalytic cycle. This conclusion agrees with different redox states of heme a3 in the K362M and E286Q mutants under aerobic steady-state turnover conditions.

Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease

Little is known about the potential for engraftment of autologous hematopoietic stem cells in human adults not subjected to myeloablative conditioning regimens. Five adult patients with the p47phox deficiency form of chronic granulomatous disease received intravenous infusions of autologous CD34+ peripheral blood stem cells (PBSCs) that had been transduced ex vivo with a recombinant retrovirus encoding normal p47phox. Although marrow conditioning was not given, functionally corrected granulocytes were detectable in peripheral blood of all five patients. Peak correction occurred 3–6 weeks after infusion and ranged from 0.004 to 0.05% of total peripheral blood granulocytes. Corrected cells were detectable for as long as 6 months after infusion in some individuals. Thus, prolonged engraftment of autologous PBSCs and continued expression of the transduced gene can occur in adults without conditioning. This trial also piloted the use of animal protein-free medium and a blood-bank-compatible closed system of gas-permeable plastic containers for culture and transduction of the PBSCs. These features enhance the safety of PBSCs directed gene therapy.

Mass spectrometric determination of dioxygen bond splitting in the “peroxy” intermediate of cytochrome c oxidase

The “peroxy” intermediate (P form) of bovine cytochrome c oxidase was prepared by reaction of the two-electron reduced mixed-valence CO complex with 18O2 after photolytic removal of CO. The water present in the reaction mixture was recovered and analyzed for 18O enrichment by mass spectrometry. It was found that approximately one oxygen atom (18O) per one equivalent of the P form was present in the bulk water. The data show that the oxygen–oxygen dioxygen bond is already broken in the P intermediate and that one oxygen atom can be readily released or exchanged with the oxygen of the solvent water.

NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines

Pulmonary neuroepithelial bodies (NEB) are widely distributed throughout the airway mucosa of human and animal lungs. Based on the observation that NEB cells have a candidate oxygen sensor enzyme complex (NADPH oxidase) and an oxygen-sensitive K+ current, it has been suggested that NEB may function as airway chemoreceptors. Here we report that mRNAs for both the hydrogen peroxide sensitive voltage gated potassium channel subunit (KH2O2) KV3.3a and membrane components of NADPH oxidase (gp91phox and p22phox) are coexpressed in the NEB cells of fetal rabbit and neonatal human lungs. Using a microfluorometry and dihydrorhodamine 123 as a probe to assess H2O2 generation, NEB cells exhibited oxidase activity under basal conditions. The oxidase in NEB cells was significantly stimulated by exposure to phorbol esther (0.1 μM) and inhibited by diphenyliodonium (5 μM). Studies using whole-cell voltage clamp showed that the K+ current of cultured fetal rabbit NEB cells exhibited inactivating properties similar to KV3.3a transcripts expressed in Xenopus oocyte model. Exposure of NEB cells to hydrogen peroxide (H2O2, the dismuted by-product of the oxidase) under normoxia resulted in an increase of the outward K+ current indicating that H2O2 could be the transmitter modulating the O2-sensitive K+ channel. Expressed mRNAs or orresponding protein products for the NADPH oxidase membrane cytochrome b as well as mRNA encoding KV3.3a were identified in small cell lung carcinoma cell lines. The studies presented here provide strong evidence for an oxidase-O2 sensitive potassium channel molecular complex operating as an O2 sensor in NEB cells, which function as chemoreceptors in airways and in NEB related tumors. Such a complex may represent an evolutionary conserved biochemical link for a membrane bound O2-signaling mechanism proposed for other cells and life forms.

Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: A di-heme active site?

Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b595 was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the α-band of heme b595. The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b595 within a few ps, pointing to a direct interaction between hemes b595 and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b595 initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b595 provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and b595 and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.

PU.1 as an essential activator for the expression of gp91phox gene in human peripheral neutrophils, monocytes, and B lymphocytes

We have reported a deficiency of a 91-kDa glycoprotein component of the phagocyte NADPH oxidase (gp91phox) in neutrophils, monocytes, and B lymphocytes of a patient with X chromosome-linked chronic granulomatous disease. Sequence analysis of his gp91phox gene revealed a single-base mutation (C → T) at position −53. Electrophoresis mobility-shift assays showed that both PU.1 and hematopoietic-associated factor 1 (HAF-1) bound to the inverted PU.1 consensus sequence centered at position −53 of the gp91phox promoter, and the mutation at position −53 strongly inhibited the binding of both factors. It was also indicated that a mutation at position −50 strongly inhibited PU.1 binding but hardly inhibited HAF-1 binding, and a mutation at position −56 had an opposite binding specificity for these factors. In transient expression assay using HEL cells, which express PU.1 and HAF-1, the mutations at positions −53 and −50 significantly reduced the gp91phox promoter activity; however, the mutation at position −56 did not affect the promoter activity. In transient cotransfection study, PU.1 dramatically activated the gp91phox promoter in Jurkat T cells, which originally contained HAF-1 but not PU.1. In addition, the single-base mutation (C → T) at position −52 that was identified in a patient with chronic granulomatous disease inhibited the binding of PU.1 to the promoter. We therefore conclude that PU.1 is an essential activator for the expression of gp91phox gene in human neutrophils, monocytes, and B lymphocytes.

The domain structure of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides

Protoporphyrinogen oxidase (EC 1–3-3–4), the 60-kDa membrane-bound flavoenzyme that catalyzes the final reaction of the common branch of the heme and chlorophyll biosynthesis pathways in plants, is the molecular target of diphenyl ether-type herbicides. It is highly resistant to proteases (trypsin, endoproteinase Glu-C, or carboxypeptidases A, B, and Y), because the protein is folded into an extremely compact form. Trypsin maps of the native purified and membrane-bound yeast protoporphyrinogen oxidase show that this basic enzyme (pI > 8.5) was cleaved at a single site under nondenaturing conditions, generating two peptides with relative molecular masses of 30,000 and 35,000. The endoproteinase Glu-C also cleaved the protein into two peptides with similar masses, and there was no additional cleavage site under mild denaturing conditions. N-terminal peptide sequence analysis of the proteolytic (trypsin and endoproteinase Glu-C) peptides showed that both cleavage sites were located in putative connecting loop between the N-terminal domain (25 kDa) with the βαβ ADP-binding fold and the C-terminal domain (35 kDa), which possibly is involved in the binding of the isoalloxazine moiety of the FAD cofactor. The peptides remained strongly associated and fully active with the Km for protoporphyrinogen and the Ki for various inhibitors, diphenyl-ethers, or diphenyleneiodonium derivatives, identical to those measured for the native enzyme. However, the enzyme activity of the peptides was much more susceptible to thermal denaturation than that of the native protein. Only the C-terminal domain of protoporphyrinogen oxidase was labeled specifically in active site-directed photoaffinity-labeling experiments. Trypsin may have caused intramolecular transfer of the labeled group to reactive components of the N-terminal domain, resulting in nonspecific labeling. We suggest that the active site of protoporphyrinogen oxidase is in the C-terminal domain of the protein, at the interface between the C- and N-terminal domains.

The Mechanism of Rhythmic Ethylene Production in Sorghum. The Role of Phytochrome B and Simulated Shading1

Mutant sorghum (Sorghum bicolor [L.] Moench) deficient in functional phytochrome B exhibits reduced photoperiodic sensitivity and constitutively expresses a shade-avoidance phenotype. Under relatively bright, high red:far-red light, ethylene production by seedlings of wild-type and phytochrome B-mutant cultivars progresses through cycles in a circadian rhythm; however, the phytochrome B mutant produces ethylene peaks with approximately 10 times the amplitude of the wild type. Time-course northern blots show that the mutant's abundance of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase mRNA SbACO2 is cyclic and is commensurate with ethylene production, and that ACC oxidase activity follows the same pattern. Both SbACO2 abundance and ACC oxidase activity in the wild-type plant are very low under this regimen. ACC levels in the two cultivars did not demonstrate fluctuations coincident with the ethylene produced. Simulated shading caused the wild-type plant to mimic the phenotype of the mutant and to produce high amplitude rhythms of ethylene evolution. The circadian feature of the ethylene cycle is conditionally present in the mutant and absent in the wild-type plant under simulated shading. SbACO2 abundance in both cultivars demonstrates a high-amplitude diurnal cycle under these conditions; however, ACC oxidase activity, although elevated, does not exhibit a clear rhythm correlated with ethylene production. ACC levels in both cultivars show fluctuations corresponding to the ethylene rhythm previously observed. It appears that at least two separate mechanisms may be involved in generating high-amplitude ethylene rhythms in sorghum, one in response to the loss of phytochrome B function and another in response to shading.

On the role of the K-proton transfer pathway in cytochrome c oxidase

Cytochrome c oxidase is a membrane-bound enzyme that catalyzes the four-electron reduction of oxygen to water. This highly exergonic reaction drives proton pumping across the membrane. One of the key questions associated with the function of cytochrome c oxidase is how the transfer of electrons and protons is coupled and how proton transfer is controlled by the enzyme. In this study we focus on the function of one of the proton transfer pathways of the R. sphaeroides enzyme, the so-called K-proton transfer pathway (containing a highly conserved Lys(I-362) residue), leading from the protein surface to the catalytic site. We have investigated the kinetics of the reaction of the reduced enzyme with oxygen in mutants of the enzyme in which a residue [Ser(I-299)] near the entry point of the pathway was modified with the use of site-directed mutagenesis. The results show that during the initial steps of oxygen reduction, electron transfer to the catalytic site (to form the “peroxy” state, Pr) requires charge compensation through the proton pathway, but no proton uptake from the bulk solution. The charge compensation is proposed to involve a movement of the K(I-362) side chain toward the binuclear center. Thus, in contrast to what has been assumed previously, the results indicate that the K-pathway is used during oxygen reduction and that K(I-362) is charged at pH ≈ 7.5. The movement of the Lys is proposed to regulate proton transfer by “shutting off” the protonic connectivity through the K-pathway after initiation of the O2 reduction chemistry. This “shutoff” prevents a short-circuit of the proton-pumping machinery of the enzyme during the subsequent reaction steps.

Saline Stress Alters the Temporal Patterns of Xylem Differentiation and Alternative Oxidase Expression in Developing Soybean Roots1

We conducted a coordinated biochemical and morphometric analysis of the effect of saline conditions on the differentiation zone of developing soybean (Glycine max L.) roots. Between d 3 and d 14 for seedlings grown in control or NaCl-supplemented medium, we studied (a) the temporal evolution of the respiratory alternative oxidase (AOX) capacity in correlation with the expression and localization of AOX protein analyzed by tissue-print immunoblotting; (b) the temporal evolution and tissue localization of a peroxidase activity involved in lignification; and (c) the structural changes, visualized by light microscopy and quantified by image digitization. The results revealed that saline stress retards primary xylem differentiation. There is a corresponding delay in the temporal pattern of AOX expression, which is consistent with the xylem-specific localization of AOX protein and the idea that this enzyme is linked to xylem development. An NaCl-induced acceleration of the development of secondary xylem was also observed. However, the temporal pattern of a peroxidase activity localized in the primary and secondary xylem was unaltered by NaCl treatment. Thus, the NaCl-stressed root was specifically affected in the temporal patterns of AOX expression and xylem development.

Molecular Characterization of the Oxalate Oxidase Involved in the Response of Barley to the Powdery Mildew Fungus1

Previously we reported that oxalate oxidase activity increases in extracts of barley (Hordeum vulgare) leaves in response to the powdery mildew fungus (Blumeria [syn. Erysiphe] graminis f.sp. hordei) and proposed this as a source of H2O2 during plant-pathogen interactions. In this paper we show that the N terminus of the major pathogen-response oxalate oxidase has a high degree of sequence identity to previously characterized germin-like oxalate oxidases. Two cDNAs were isolated, pHvOxOa, which represents this major enzyme, and pHvOxOb', representing a closely related enzyme. Our data suggest the presence of only two oxalate oxidase genes in the barley genome, i.e. a gene encoding HvOxOa, which possibly exists in several copies, and a single-copy gene encoding HvOxOb. The use of 3′ end gene-specific probes has allowed us to demonstrate that the HvOxOa transcript accumulates to 6 times the level of the HvOxOb transcript in response to the powdery mildew fungus. The transcripts were detected in both compatible and incompatible interactions with a similar accumulation pattern. The oxalate oxidase is found exclusively in the leaf mesophyll, where it is cell wall located. A model for a signal transduction pathway in which oxalate oxidase plays a central role is proposed for the regulation of the hypersensitive response.