Pr State Research Articles

The Crystal Structures of the N-terminal Photosensory Core Module of Agrobacterium Phytochrome Agp1 as Parallel and Anti-parallel Dimers

Agp1 is a canonical biliverdin-binding bacteriophytochrome from the soil bacterium Agrobacterium fabrum that acts as a light-regulated histidine kinase. Crystal structures of the photosensory core modules (PCMs) of homologous phytochromes have provided a consistent picture of the structural changes that these proteins undergo during photoconversion between the parent red light-absorbing state (Pr) and the far-red light-absorbing state (Pfr). These changes include secondary structure rearrangements in the so-called tongue of the phytochrome-specific (PHY) domain and structural rearrangements within the long α-helix that connects the cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) and the PHY domains. We present the crystal structures of the PCM of Agp1 at 2.70 Å resolution and of a surface-engineered mutant of this PCM at 1.85 Å resolution in the dark-adapted Pr states. Whereas in the mutant structure the dimer subunits are in anti-parallel orientation, the wild-type structure contains parallel subunits. The relative orientations between the PAS-GAF bidomain and the PHY domain are different in the two structures, due to movement involving two hinge regions in the GAF-PHY connecting α-helix and the tongue, indicating pronounced structural flexibility that may give rise to a dynamic Pr state. The resolution of the mutant structure enabled us to detect a sterically strained conformation of the chromophore at ring A that we attribute to the tight interaction with Pro-461 of the conserved PRXSF motif in the tongue. Based on this observation and on data from mutants where residues in the tongue region were replaced by alanine, we discuss the crucial roles of those residues in Pr-to-Pfr photoconversion.

Structures of the photosensory core module of bacteriophytochrome Agp1 from two crystal forms reveal plasticity of the Pr state

Structures of the photosensory core module of bacteriophytochrome Agp1 from two crystal forms reveal plasticity of the Pr state

Structure of the Full-Length Bacteriophytochrome from the Plant Pathogen Xanthomonas campestris Provides Clues to its Long-Range Signaling Mechanism

Phytochromes constitute a major superfamily of light-sensing proteins that are reversibly photoconverted between a red-absorbing (Pr) and a far-red-absorbing (Pfr) state. Bacteriophytochromes (BphPs) are found among photosynthetic and non-photosynthetic bacteria, including pathogens. To date, several BphPs have been biophysically characterized. However, it is still not fully understood how structural changes are propagated from the photosensory module to the output module during the signal transduction event. Most phytochromes share a common architecture consisting of an N-terminal photosensor that includes the PAS2–GAF–PHY domain triad and a C-terminal variable output module. Here we present the crystal structure of the full-length BphP from the plant pathogen Xanthomonas campestris pv. campestris (XccBphP) bearing its photosensor and its complete output module, a PAS9 domain. In the crystals, the protein was found to be in the Pr state, whereas diffraction data together with resonance Raman spectroscopic and theoretical results indicate a ZZZssa and a ZZEssa chromophore configuration corresponding to a mixture of Pr and Meta-R state, the precursor of Pfr. The XccBphP quaternary assembly reveals a head-to-head dimer in which the output module contributes to the helical dimer interface. The photosensor, which is shown to be a bathy-like BphP, is influenced in its dark reactions by the output module. Our structural analyses suggest that the photoconversion between the Pr and Pfr states in the full-length XccBphP may involve changes in the relative positioning of the output module. This work contributes to understand the light-induced structural changes propagated from the photosensor to the output modules in phytochrome signaling.

Spectroscopic Investigation on the Primary Photoreaction of Bathy Phytochrome Agp2-Pr of Agrobacterium fabrum: Isomerization in a pH-dependent H-bond Network.

Bathy phytochrome Agp2 from Agrobacterium fabrum exhibits an unusually low pKa =7.6 in the Pr state in contrast to a pKa >11 in the Pfr state, indicating a pH-dependent charge distribution and H-bond network in the Pr chromophore binding pocket around neutral pH. Here, we report on ultrafast UV/Vis absorption spectroscopy of the primary Pr photoisomerization of Agp2 at pH 6 and pH 9 and upon H2 O/D2 O buffer exchange. The triexponential Pr kinetics slows down at increased pH and pronounced pH-dependent kinetic isotope effects are observed. The results on the Pr photoreaction suggest: 1) component-wise hindered dynamics on the chromophore excited-state potential energy surface at high pH and 2) proton translocation processes either via single-proton transfer or via significant reorganization of H-bond networks. Both effects reflect the interplay between the pH-dependent charge distribution in the Pr chromophore binding pocket on the one hand and chromophore excitation and its Z→E isomerization on the other hand.

DETERMINAÇÃO DA ÉPOCA DE DESBASTE PELA ANÁLISE DENDROCRONOLÓGICA E MORFOMÉTRICA DE<i> Ocotea porosa</i> (Nees & Mart.) Barroso EM POVOAMENTO NÃO MANEJADO

http://dx.doi.org/10.5902/1980509819620Plantios comerciais de Ocotea porosa disponíveis para estudo são raros e por esse motivo devem ser usados como fonte de informação para se estabelecer o potencial de manejo da espécie. A recuperação do histórico do crescimento desses povoamentos para estruturação dos planos de manejo é possível com o uso da técnica da dendrocronologia e a análise de dados dendrométricos e morfométricos. O objetivo desse trabalho foi utilizar ferramentas de análise de povoamentos florestais aliadas aos estudos dendrocronológicos, para recuperar e sistematizar o desenvolvimento de povoamento não manejado. O trabalho foi conduzido em um povoamento não manejado de imbuia, com 44 anos, em Rio Negro - PR, na Estação Experimental da UFPR. Inferências do crescimento recuperado por dendrocronologia, e competição pela aplicação de metodologias como Grau de Esbeltez e de projeção de copa mostraram resultados compatíveis na análise do povoamento, indicando necessidade de primeiro desbaste próximo aos 12 anos. Em função do pequeno número de árvores amostradas, os resultados não permitem generalizações definitivas, entretanto, são consistentes para estabelecer um protocolo para análise de povoamentos não manejados de Ocotea porosa, assim como de outras espécies.

A Red/Green Cyanobacteriochrome Sustains Its Color Despite a Change in the Bilin Chromophore's Protonation State.

The second GAF domain of AnPixJ, AnPixJg2, a bilin-binding protein from the cyanobacterium Anabaena PCC 7120, undergoes a photoinduced interconversion between a red-absorbing state, Pr, and a green-absorbing state, Pg. Combining ultraviolet-vis (UV-vis), infrared, resonance Raman (RR), and magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, we have studied this cyanobacteriochrome (CBCR) assembled with phycocyanobilin (PCB) either in vivo or in vitro. In both assembly routes, the spectroscopic data of the Pr state reveal nearly identical chromophore structures with a protonated (cationic) bilin. However, unlike the native (in vivo assembly) Pg photoproduct, in which the bilin retains protonation, the Pg generated from the in vitro-assembled AnPixJg2 harbors a deprotonated (neutral) bilin chromophore at pH 7.8. IR difference spectroscopy further reveals the transfer of a proton from the bilin to a side-chain carboxylate on an amino acid, probably Asp291. Besides the change in protonation state, the bilin structure is very similar in the in vitro- and in vivo-assembled Pg photoproducts. The chromophore of the in vitro Pg becomes protonated when the pH is increased to 10, presumably because of a partial reversal of protein misfolding. Most remarkably, the electronic transitions remain unchanged and are very similar to those of the native Pg. Thus, bilin protonation is not a key parameter for controlling the energies of the electronic transitions in AnPixJg2. Possible alternative molecular mechanisms for color tuning are discussed.

A Curatorial Turn in Policy Development? Managing the Changing Nature of Policymaking Subject to Mediatisation

A Curatorial Turn in Policy Development? Managing the Changing Nature of Policymaking Subject to Mediatisation

NMR chemical shift pattern changed by ammonium sulfate precipitation in cyanobacterial phytochrome Cph1.

Phytochromes are dimeric biliprotein photoreceptors exhibiting characteristic red/far-red photocycles. Full-length cyanobacterial phytochrome Cph1 from Synechocystis 6803 is soluble initially but tends to aggregate in a concentration-dependent manner, hampering attempts to solve the structure using NMR and crystallization methods. Otherwise, the Cph1 sensory module (Cph1Δ2), photochemically indistinguishable from the native protein and used extensively in structural and other studies, can be purified to homogeneity in >10 mg amounts at mM concentrations quite easily. Bulk precipitation of full-length Cph1 by ammonium sulfate (AmS) was expected to allow us to produce samples for solid-state magic-angle spinning (MAS) NMR from dilute solutions before significant aggregation began. It was not clear, however, what effects the process of partial dehydration might have on the molecular structure. Here we test this by running solid-state MAS NMR experiments on AmS-precipitated Cph1Δ2 in its red-absorbing Pr state carrying uniformly 13C/15N-labeled phycocyanobilin (PCB) chromophore. 2D 13C–13C correlation experiments allowed a complete assignment of 13C responses of the chromophore. Upon precipitation, 13C chemical shifts for most of PCB carbons move upfield, in which we found major changes for C4 and C6 atoms associated with the A-ring positioning. Further, the broad spectral lines seen in the AmS 13C spectrum reflect primarily the extensive inhomogeneous broadening presumably due to an increase in the distribution of conformational states in the protein, in which less free water is available to partake in the hydration shells. Our data suggest that the effect of dehydration process indeed leads to changes of electronic structure of the bilin chromophore and a decrease in its mobility within the binding pocket, but not restricted to the protein surface. The extent of the changes induced differs from the freezing process of the solution samples routinely used in previous MAS NMR and crystallographic studies. AmS precipitation might nevertheless provide useful protein structure/functional information for full-length Cph1 in cases where neither X-ray crystallography nor conventional NMR methods are available.

Color Tuning in Red/Green Cyanobacteriochrome AnPixJ: Photoisomerization at C15 Causes an Excited-State Destabilization.

Cyanobacteriochromes (CBCRs) are cyanobacterial phytochrome-like photoreceptors that carry a single or several GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains in a repetitive manner. Unlike phytochromes that photoswitch between red-absorbing 15Z Pr and far-red-absorbing 15E Pfr states, CBCRs exhibit a much wider spectral activity. One of the best-characterized CBCRs, the phototaxis regulator PixJ of Anabaena sp. PCC 7120, AnPixJ can adopt two thermally stable photoreversible states, a red-absorbing dark state (Pr) and a green-absorbing photoproduct (Pg). Cross-polarization magic-angle spinning (CP/MAS) NMR spectroscopy on AnPixJ assembled in vitro with uniformly (13)C- and (15)N-labeled phycocyanobilin (PCB) chromophore identifies changes of the electronic structure of the chromophore between the two states. Results are compared with the data from red- and far-red-absorbing forms of the complete sensory module of cyanobacterial phytochrome Cph1 aiming at a conceptual understanding of the distinct photoproduct (Pg vs Pfr) absorbances upon Pr photoconversion. The PCB chromophore in the Pr state of both photosensors exhibits very similar spectral features. The photoconversion of Cph1 and the red/green switching AnPixJ C15-Z/E photoisomerization result in a very similar chemical-shift difference (Δδ) pattern having, however, opposite sign. The persistence of this pattern confirms the identity of the photochemical isomerization process, while the difference in its sign demonstrates that the same electronic factors drive into opposite direction. It is proposed that the LUMO energy of the 15E photoproduct is stabilized in Cph1 but destabilized in AnPixJ leading to opposite color shifts upon phototransformation.

Unusual Temperature Dependence of Magnetization and Possible Magnetic Noncollinearity in Tm and Pr Clusters.

Rare-earth metals in their bulk form possess rather similar crystallographic structures, which is due to the very similar features of their outer electronic states. On the other hand, their magnetic properties are of rich variety, which is related to the specific form of the indirect magnetic exchange interaction between the inner electronic shells. In cluster form, this interplay may lead to very unusual magnetic structures. Here we show how the magnetic moments vary with size and temperature in Tm and Pr clusters. While in Pr clusters clear atom-by-atom oscillations indicate antiferromagnetic ordering, smooth variation and anomalous temperature behavior in Tm is representative for an essentially non-collinear spin arrangement. Their electric behavior is also very different, with a metallic-like behavior of Pr and localized electronic states in Tm.

X-ray Radiation Induces Deprotonation of the Bilin Chromophore in Crystalline D. radiodurans Phytochrome

We report that in the red light-absorbing (Pr) state, the bilin chromophore of the Deinococcus radiodurans proteobacterial phytochrome (DrBphP) is hypersensitive to X-ray photons used in typical synchrotron X-ray protein crystallography experiments. This causes the otherwise fully protonated chromophore to deprotonate without additional major structural changes. These results have major implications for our understanding of the structural and chemical characteristics of the resting and intermediate states of phytochromes and other photoreceptor proteins.

Connection between Absorption Properties and Conformational Changes in Deinococcus radiodurans Phytochrome

Phytochromes consist of several protein domains and a linear tetrapyrrole molecule, which interact as a red-light-sensing system. In this study, size-exclusion chromatography and light-scattering techniques are combined with UV-vis spectroscopy to investigate light-induced changes in dimeric Deinococcus radiodurans bacterial phytochrome (DrBphP) and its subdomains. The photosensory unit (DrCBD-PHY) shows an unusually stable Pfr state with minimal dark reversion, whereas the histidine kinase (HK) domain facilitates dark reversion to the resting state. Size-exclusion chromatography reveals that all phytochrome fragments remain as dimers in the illuminated state and dark state. Still, the elution profiles of all phytochrome fragments differ between the illuminated and dark states. The differences are observed reliably only when the whole UV-vis spectrum is characterized along the elution profile and show more Pfr-state characteristics at later elution volumes in DrBphP and DrCBD-PHY fragments. This implies that the PHY domain has an important role in amplifying and relaying light-induced conformational changes to the HK domain. In the illuminated state, the HK domain appears partially unfolded and prone to form oligomers. The oligomerization of DrBphP can be diminished by converting the molecule back to the resting Pr state by using far-red light.

FTIR Spectroscopy Revealing Light-Dependent Refolding of the Conserved Tongue Region of Bacteriophytochrome.

Bacteriophytochromes (BphPs) constitute a class of photosensory proteins that toggle between Pr and Pfr functional states through absorption of red and far-red light. The photosensory core of BphPs is composed of PAS, GAF, and PHY domains. Here, we apply FTIR spectroscopy to investigate changes in the secondary structure of Rhodopseudomonas palustris BphP2 (RpBphP2) upon Pr to Pfr photoconversion. Our results indicate conversion from a β-sheet to an α-helical element in the so-called tongue region of the PHY domain, consistent with recent X-ray structures of Deinococcus radiodurans DrBphP in dark and light states (TakalaH.; et al. Nature2014, 509, 245−24824776794). A conserved Asp in the GAF domain that noncovalently connects with the PHY domain and a conserved Pro in the tongue region of the PHY domain are essential for the β-sheet-to-α-helix conversion.

Crystal structure of the photosensing module from a red/far-red light-absorbing plant phytochrome.

Many aspects of plant photomorphogenesis are controlled by the phytochrome (Phy) family of bilin-containing photoreceptors that detect red and far-red light by photointerconversion between a dark-adapted Pr state and a photoactivated Pfr state. Whereas 3D models of prokaryotic Phys are available, models of their plant counterparts have remained elusive. Here, we present the crystal structure of the photosensing module (PSM) from a seed plant Phy in the Pr state using the PhyB isoform from Arabidopsis thaliana. The PhyB PSM crystallized as a head-to-head dimer with strong structural homology to its bacterial relatives, including a 5(Z)syn, 10(Z)syn, 15(Z)anti configuration of the phytochromobilin chromophore buried within the cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain, and a well-ordered hairpin protruding from the Phy-specific domain toward the bilin pocket. However, its Per/Arnt/Sim (PAS) domain, knot region, and helical spine show distinct structural differences potentially important to signaling. Included is an elongated helical spine, an extended β-sheet connecting the GAF domain and hairpin stem, and unique interactions between the region upstream of the PAS domain knot and the bilin A and B pyrrole rings. Comparisons of this structure with those from bacterial Phys combined with mutagenic studies support a toggle model for photoconversion that engages multiple features within the PSM to stabilize the Pr and Pfr end states after rotation of the D pyrrole ring. Taken together, this Arabidopsis PhyB structure should enable molecular insights into plant Phy signaling and provide an essential scaffold to redesign their activities for agricultural benefit and as optogenetic reagents.

Prevalence and genotyping of pathogenic Escherichia coli on carcasses of pigs slaughtered in commercial slaughterhouses in southern region of Brazil

A carne suína representa importante fonte de proteína animal para o mundo, porem vem sendo associada a surtos de toxinfecção alimentar. Uma das causas destes surtos é a contaminação por Escherichia coli , encontrada no trato intestinal e ambiente dos suínos abatidos para produção de carnes in natura e industrializadas. No contexto de segurança alimentar, os surtos por Escherichia coli produtoras de toxina Shiga (STEC) são os melhores documentados. Diversos surtos causados por ingestão de alimentos de origem animal foram documentados. Contudo, no Brasil, existem poucos dados sobre a ocorrência deste patógenos em suínos e, consequentemente, na carne de porco. O objetivo deste trabalho foi quantificar a contaminação por E. coli de carcaças suínas abatidos em abatedouros comerciais localizados nos estados da Região Sul do Brasil, e identificar por PCR a presença de E. coli produtora das toxinas Shiga e Intimina. Foram realizados swabs de 272 carcaças suínas em abatedouros localizados nos estados do RS, SC e PR. Foram identificadas contaminações por E. coli em 25 carcaças, sendo 20 no abatedouro do RS e cinco no abatedouro paranaense. Das amostras positivas foram extraídos DNA para genotipagem por PCR. Nenhuma amostra apresentou o gene stx1, porém o gene eaeA foi identificado em 13 amostras, nas diferentes regiões da carcaça. A técnica de PCR pode ser uma ferramenta útil no rastreamento da contaminação bacteriana ao longo dos processos do abatedouro, podendo auxiliar na redução da incidência dos casos de toxinfecções alimentares causadas por E. coli e outros microorganismos.

Structure of the Cyanobacterial Phytochrome 2 Photosensor Implies a Tryptophan Switch for Phytochrome Signaling

Phytochromes are highly versatile photoreceptors, which occur ubiquitously in plants as well as in many light-responsive microorganisms. Here, photosynthetic cyanobacteria utilize up to three different phytochrome architectures, where only the plant-like and the single-domain cyanobacteriochromes are structurally characterized so far. Cph2 represents a third group in Synechocystis species and affects their capability of phototaxis by controlling c-di-GMP synthesis and degradation. The 2.6-Å crystal structure of its red/far-red responsive photosensory module in the Pr state reveals a tandem-GAF bidomain that lacks the figure-of-eight knot of the plant/cph1 subfamily. Its covalently attached phycocyanobilin chromophore adopts a highly tilted ZZZssa conformation with a novel set of interactions between its propionates and the GAF1 domain. The tongue-like protrusion from the GAF2 domain interacts with the GAF1-bound chromophore via its conserved PRXSF, WXE, and W(G/A)G motifs. Mutagenesis showed that the integrity of the tongue is indispensable for Pr → Pfr photoconversion and involves a swap of the motifs' tryptophans within the tongue-GAF1 interface. This "Trp switch" is supposed to be a crucial element for the photochromicity of all multidomain phytochromes.

Kinetic and Thermodynamic Analysis of the Light-induced Processes in Plant and Cyanobacterial Phytochromes

The light-induced processes of the biological photoreceptor phytochrome (recombinant phyA of oat and recombinant CphA from the cyanobacterium Tolypothrix PCC7601) have been investigated in a time-resolved manner in the temperature range from 0 to 30°C. Both proteins were heterologously expressed and assembled in vitro with phycocyanobilin. The Pr state of plant phytochrome phyA is converted to the Pfr state after formation of four intermediates with an overall quantum yield of ∼18%. The reversal reaction (Pfr-to-Pr) shows several intermediates, all of which, even the first detectable one, exhibit already all spectral features of the Pr state. The canonical phytochrome CphA from Tolypothrix showed a similar intermediate sequence as its plant ortholog. Whereas the kinetics for the forward reaction (Pr-to-Pfr) was nearly identical for both proteins, the reverse process (Pr formation) in the cyanobacterial phytochrome was slower by a factor of three. As found for the Pfr-to-Pr intermediates in the plant protein, also in CphA all detectable intermediates showed the spectral features of the Pr form. For both phytochromes, activation parameters for both the forward and the backward reaction pathways were determined.

Photoconversion Mechanism of the Second GAF Domain of Cyanobacteriochrome AnPixJ and the Cofactor Structure of Its Green-Absorbing State

Cyanobacteriochromes are members of the phytochrome superfamily. In contrast to classical phytochromes, these small photosensors display a considerable variability of electronic absorption maxima. We have studied the light-induced conversions of the second GAF domain of AnPixJ, AnPixJg2, a phycocyanobilin-binding protein from the cyanobacterium Anabaena PCC 7120, using low-temperature resonance Raman spectroscopy combined with molecular dynamics simulations. AnPixJg2 is formed biosynthetically as a red-absorbing form (Pr) and can be photoconverted into a green-absorbing form (Pg). Forward and backward phototransformations involve the same reaction sequences and intermediates of similar cofactor structures as the corresponding processes in canonical phytochromes, including a transient cofactor deprotonation. Whereas the cofactor of the Pr state shows far-reaching similarities to the Pr states of classical phytochromes, the Pg form displays significant upshifts of the methine bridge stretching frequencies concomitant to the hypsochromically shifted absorption maximum. However, the cofactor in Pg is protonated and adopts a conformation very similar to the Pfr state of classical phytochromes. The spectral differences are probably related to an increased solvent accessibility of the chromophore which may reduce the π-electron delocalization in the phycocyanobilin and thus raise the energies of the first electronic transition and the methine bridge stretching modes. Molecular dynamics simulations suggest that the Z → E photoisomerization of the chromophore at the C-D methine bridge alters the interactions with the nearby Trp90 which in turn may act as a gate, allowing the influx of water molecules into the chromophore pocket. Such a mechanism of color tuning AnPixJg2 is unique among the cyanobacteriochromes studied so far.

Structural and sequence analyses of an unusual bacteriophytochrome from R. palustris

Bacteriophytochromes (Bphs) are bacterial red‐light photoreceptors found in photosynthetic and non‐photosynthetic bacteria. Bphs undergo reversible photoconversion between two distinct red (Pr) and far‐red (Pfr) light absorbing states. An organic cofactor, biliverdin (BV) is required for Pr/Pfr photoconversion. Bphs are composed of a photosensory core (PC) including PAS, GAF and PHY domains and an effector domain, usually a histidine kinase. We report on the preliminary crystal structure of the unusual Bph from Rhodopseudomonas palustris, RpBphP3 (P3) PC bound to BV in the Pr state. Unlike classical Bphs, P3 photoconverts between Pr and near‐red (Pnr) states. P3 crystals (30–60 □m in all dimensions) yielded diffraction at 100 K to a maximum resolution of 2.8 Å. P3 crystals are in P212121 space group with unit cell dimensions of a=155.92 Å, b=185.32 Å, and c=199.53 Å. The initial P3 structure was determined with the molecular replacement (MR) method implemented in HKL3000 using the crystal structure of P3 lacking PHY domain (PDB ID code 2OOL) as search model. P3 crystallized as a dimer, which is physiologically relevant oligomeric state of the protein. Informed by structural and sequence analyses, we identify amino acids in the BV‐binding pocket of P3 and at the dimer interface that play a role in the unusual photochemistry and fluorescence of this protein.

Spectral Identification of Intermediates Generated during the Reaction of Dioxygen with the Wild-Type and EQ(I-286) Mutant of Rhodobacter sphaeroides Cytochrome c Oxidase

Cytochrome c oxidase from Rhodobacter sphaeroides is frequently used to model the more complex mitochondrial enzyme. The O(2) reduction in both enzymes is generally described by a unidirectional mechanism involving the sequential formation of the ferrous-oxy complex (compound A), the P(R) state, the oxyferryl F form, and the oxidized state. In this study we investigated the reaction of dioxygen with the wild-type reduced R. sphaeroides cytochrome oxidase and the EQ(I-286) mutant using the CO flow-flash technique. Singular value decomposition and multiexponential fitting of the time-resolved optical absorption difference spectra showed that three apparent lifetimes, 18 μs, 53 μs, and 1.3 ms, are sufficient to fit the kinetics of the O(2) reaction of the wild-type enzyme. A comparison of the experimental intermediate spectra with the corresponding intermediate spectra of the bovine enzyme revealed that P(R) is not present in the reaction mechanism of the wild-type R. sphaeroides aa(3). Transient absorbance changes at 440 and 610 nm support this conclusion. For the EQ(I-286) mutant, in which a key glutamic residue in the D proton pathway is replaced by glutamine, two lifetimes, 16 and 108 μs, were observed. A spectral analysis of the intermediates shows that the O(2) reaction in the EQ(I-286) mutant terminates at the P(R) state, with 70% of heme a becoming oxidized. These results indicate significant differences in the kinetics of O(2) reduction between the bovine and wild-type R. sphaeroides aa(3) oxidases, which may arise from differences in the relative rates of internal electron and proton movements in the two enzymes.