Utilization of blue-green light by chlorosomes from the photosynthetic bacterium Chloroflexus aurantiacus: Ultrafast excitation energy conversion and transfer

Chlorosomes of photosynthetic green bacteria are unique molecular assemblies providing efficient light harvesting followed by multi-step transfer of excitation energy to reaction centers. In each chlorosome, 10⁴–10⁵ bacteriochlorophyll (BChl) c/d/e molecules are organized by self-assembly into high-ordered aggregates. We studied the early-time dynamics of the excitation energy flow and energy conversion in chlorosomes isolated from Chloroflexus (Cfx.) aurantiacus bacteria by pump-probe spectroscopy with 30-fs temporal resolution at room temperature. Both the S₂ state of carotenoids (Cars) and the Soret states of BChl c were excited at ~490 nm, and absorption changes were probed at 400–900 nm. A global analysis of spectroscopy data revealed that the excitation energy transfer (EET) from Cars to BChl c aggregates occurred within ~100 fs, and the Soret → Q energy conversion in BChl c occurred faster within ~40 fs. This conclusion was confirmed by a detailed comparison of the early exciton dynamics in chlorosomes with different content of Cars. These processes are accompanied by excitonic and vibrational relaxation within 100–270 fs. The well-known EET from BChl c to the baseplate BChl a proceeded on a ps time-scale. We showed that the S₁ state of Cars does not participate in EET. We discussed the possible presence (or absence) of an intermediate state that might mediates the Soret → Q_y internal conversion in chlorosomal BChl c. We discussed a possible relationship between the observed exciton dynamics and the structural heterogeneity of chlorosomes.     

Identification of the Intermediate Charge-Separated State PβL in a Leucine M214 to Histidine Mutant of the Rhodobacter sphaeroides Reaction Center Using Femtosecond Midinfrared Spectroscopy

Energy and electron transfer in a Leu M214 to His (LM214H) mutant of the Rhodobacter sphaeroides reaction center (RC) were investigated by applying time-resolved visible pump/midinfrared probe spectroscopy at room temperature. This mutant replacement of the Leu at position M214 resulted in the incorporation of a bacteriochlorophyll (BChl) in place of the native bacteriopheophytin in the L-branch of cofactors (denoted β_L). Purified LM214H RCs were excited at 600 nm (unselective excitation), at 800 nm (direct excitation of the monomeric BChl cofactors B_L and B_M), and at 860 nm (direct excitation of the primary donor (P) BChl pair (P_L/P_M)). Absorption changes associated with carbonyl (C=O) stretch vibrational modes (9-keto, 10a-ester, and 2a-acetyl) of the cofactors and of the protein were recorded in the region between 1600 cm⁻¹ and 1770 cm⁻¹, and the data were subjected to both a sequential analysis and a simultaneous target analysis. After photoexcitation of the LM214H RC, P^∗ decayed on a timescale of ∼6.3 ps to P⁺B_L⁻. The decay of P⁺B_L⁻ occurred with a lifetime of ∼2 ps, ∼3 times slower than that observed in wild-type and R-26 RCs (∼0.7 ps). Further electron transfer to the β_L BChl resulted in formation of the P⁺β_L⁻ state, and its infrared absorbance difference spectrum is reported for the first time, to our knowledge. The fs midinfrared spectra of P⁺B_L⁻ and P⁺β_L⁻ showed clear differences related to the different environments of the two BChls in the mutant RC.     

Kinetics of absorbance and anisotropy upon excited state relaxation in the reaction center core complex of a green sulfur bacterium

Properties of the excited states in reaction center core (RCC) complexes of the green sulfur bacterium Prosthecochloris aestuarii were studied by means of femtosecond time-resolved isotropic and anisotropic absorption difference spectroscopy at 275 K. Selective excitation of the different transitions of the complex resulted in the rapid establishment of a thermal equilibrium. At about 1 ps after excitation, the energy was located at the lowest energy transition, BChl a 835. Time constants varying between 0.26 and 0.46 ps were observed for the energy transfer steps leading to this equilibrium. These transfer steps were also reflected in changes in polarization. Our measurements indicate that downhill energy transfer towards excited BChl a 835 occurs via the energetically higher spectral forms BChl a 809 and BChl a 820. Low values of the anisotropy of about 0.07 were found in the ‘two-color’ measurements at 820 and 835 nm upon excitation at 800 nm, whereas the ‘one-color’ kinetics showed much higher anisotropies. Charge separation occurred with a time constant varying between 20 and 30 ps. This revised version was published online in June 2006 with corrections to the Cover Date.     

Excitation energy transfer and trapping dynamics in the core complex of the filamentous photosynthetic bacterium Roseiflexus castenholzii

The light-harvesting core complex of the thermophilic filamentous anoxygenic phototrophic bacterium Roseiflexus castenholzii is intrinsic to the cytoplasmic membrane and intimately bound to the reaction center (RC). Using ultrafast transient absorption and time-resolved fluorescence spectroscopy with selective excitation, energy transfer, and trapping dynamics in the core complex have been investigated at room temperature in both open and closed RCs. Results presented in this report revealed that the excited energy transfer from the BChl 800 to the BChl 880 band of the antenna takes about 2?ps independent of the trapping by the RC. The time constants for excitation quenching in the core antenna BChl 880 by open and closed RCs were found to be 60 and 210?ps, respectively. Assuming that the light harvesting complex is generally similar to LH1 of purple bacteria, the possible structural and functional aspects of this unique antenna complex are discussed. The results show that the core complex of Roseiflexus castenholzii contains characteristics of both purple bacteria and Chloroflexus aurantiacus.     

Properties of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> photosynthetic reaction center with double amino acid substitution I(L177)H+H(M182)L

Histidine M182 in the reaction center (RC) of Rhodobacter sphaeroides serves as the fifth ligand of the bacterio-chlorophyll (BChl) B_B Mg atom. When this His is substituted by an amino acid that is not able to coordinate Mg, bacterio-pheophytin appears in the B_B binding site instead of BChl (Katilius, E., et al. (1999) J. Phys. Chem. B, 103, 7386–7389). We have shown that in the presence of the additional mutation I(L177)H the coordination of the BChl B_B Mg atom in the double mutant I(L177)H+H(M182)L RC still remains. Changes in the double mutant RC absorption spectrum attributed to BChl absorption suggest that BChl B_B Mg atom axial ligation might be realized not from the usual α-side of the BChl macrocycle, but from the opposite, β-side. Weaker coordination of BChl B_B Mg atom compared to the other mutant RC BChl molecules suggests that not an amino acid residue but a water molecule might be a possible ligand. The results are discussed in the light of the structural changes that occurred in the RC upon Ile/His substitution in the L177 position.     

Excitation dynamics of two spectral forms of the core complexes from photosynthetic bacterium Thermochromatium tepidum

The intact core antenna-reaction center (LH1-RC) core complex of thermophilic photosynthetic bacterium Thermochromatium (Tch.) tepidum is peculiar in its long-wavelength LH1-Q_y absorption (915 nm). We have attempted comparative studies on the excitation dynamics of bacteriochlorophyll (BChl) and carotenoid (Car) between the intact core complex and the EDTA-treated one with the Q_y absorption at 889 nm. For both spectral forms, the overall Car-to-BChl excitation energy transfer efficiency is determined to be ∼20%, which is considerably lower than the reported values, e.g., ∼35%, for other photosynthetic purple bacteria containing the same kind of Car (spirilloxanthin). The RC trapping time constants are found to be 50∼60 ps (170∼200 ps) for RC in open (closed) state irrespective to the spectral forms and the wavelengths of Q_y excitation. Despite the low-energy LH1-Q_y absorption, the RC trapping time are comparable to those reported for other photosynthetic bacteria with normal LH1-Q_y absorption at 880 nm. Selective excitation to Car results in distinct differences in the Q_y-bleaching dynamics between the two different spectral forms. This, together with the Car band-shift signals in response to Q_y excitation, reveals the presence of two major groups of BChls in the LH1 of Tch. tepidum with a spectral heterogeneity of ∼240 cm⁻¹, as well as an alteration in BChl-Car geometry in the 889-nm preparation with respect to the native one.     

Excitation energy transfer in chlorosomes of Chlorobium phaeobacteroides strain CL1401: the role of carotenoids

The role of carotenoids in chlorosomes of the green sulfur bacterium Chlorobium phaeobacteroides, containing bacteriochlorophyll (BChl) e and the carotenoid (Car) isorenieratene as main pigments, was studied by steady-state fluorescence excitation, picosecond single-photon timing and femtosecond transient absorption (TA) spectroscopy. In order to obtain information about energy transfer from Cars in this photosynthetic light-harvesting antenna with high spectral overlap between Cars and BChls, Car-depleted chlorosomes, obtained by inhibition of Car biosynthesis by 2-hydroxybiphenyl, were employed in a comparative study with control chlorosomes. Excitation spectra measured at room temperature give an efficiency of 60–70% for the excitation energy transfer from Cars to BChls in control chlorosomes. Femtosecond TA measurements enabled an identification of the excited state absorption band of Cars and the lifetime of their S₁ state was determined to be 10 ps. Based on this lifetime, we concluded that the involvement of this state in energy transfer is unlikely. Furthermore, evidence was obtained for the presence of an ultrafast (>100 fs) energy transfer process from the S₂ state of Cars to BChls in control chlorosomes. Using two time-resolved techniques, we further found that the absence of Cars leads to overall slower decay kinetics probed within the Q_y band of BChl e aggregates, and that two time constants are generally required to describe energy transfer from aggregated BChl e to baseplate BChl a.This revised version was published online in October 2005 with corrections to the Cover Date.     

Ultrafast time-resolved spectroscopy of the light-harvesting complex 2 (LH2) from the photosynthetic bacterium <Emphasis Type="Italic">Thermochromatium tepidum</Emphasis>

The light-harvesting complex 2 from the thermophilic purple bacterium Thermochromatium tepidum was purified and studied by steady-state absorption and fluorescence, sub-nanosecond-time-resolved fluorescence and femtosecond time-resolved transient absorption spectroscopy. The measurements were performed at room temperature and at 10 K. The combination of both ultrafast and steady-state optical spectroscopy methods at ambient and cryogenic temperatures allowed the detailed study of carotenoid (Car)-to-bacteriochlorophyll (BChl) as well BChl-to-BChl excitation energy transfer in the complex. The studies show that the dominant Cars rhodopin (N = 11) and spirilloxanthin (N = 13) do not play a significant role as supportive energy donors for BChl a. This is related with their photophysical properties regulated by long π-electron conjugation. On the other hand, such properties favor some of the Cars, particularly spirilloxanthin (N = 13) to play the role of the direct quencher of the excited singlet state of BChl.     

Fast cyclic electron transport around photosystem I in leaves under far-red light: a proton-uncoupled pathway?

Fast cyclic electron transport (CET) around photosystem I (PS I) was observed in sunflower (Helianthus annuus L.) leaves under intense far-red light (FRL) of up to 200 μmol quanta m⁻² s⁻¹. The electron transport rate (ETR) through PS I was found from the FRL-dark transmittance change at 810 and 950 nm, which was deconvoluted into redox states and pool sizes of P700, plastocyanin (PC) and cytochrome f (Cyt f). PC and P700 were in redox equilibrium with K _e = 35 (ΔE _m = 90 mV). PS II ETR was based on O₂ evolution. CET [(PS I ETR) − (PS II ETR)] increased to 50–70 μmol e⁻ m⁻² s⁻¹ when linear electron transport (LET) under FRL was limited to 5 μmol e⁻ m⁻² s⁻¹ in a gas phase containing 20–40 μmol CO₂ mol⁻¹ and 20 μmol O₂ mol⁻¹. Under these conditions, pulse-saturated fluorescence yield F _m was non-photochemically quenched; however, F _m was similarly quenched when LET was driven by low green or white light, which energetically precluded the possibility for active CET. We suggest that under FRL, CET is rather not coupled to transmembrane proton translocation than the CET-coupled protons are short-circuited via proton channels regulated to open at high ΔpH. A kinetic analysis of CET electron donors and acceptors suggests the CET pathway is that of the reversed Q-cycle: Fd → (FNR) → Cyt c_n → Cyt b_h → Cyt b_l → Rieske FeS → Cyt f → PC → P700 →→ Fd. CET is activated when PQH₂ oxidation is opposed by high ΔpH, and ferredoxin (Fd) is reduced due to low availability of e⁻ acceptors. The physiological significance of CET may be photoprotective, as CET may be regarded as a mechanism of energy dissipation under stress conditions.     

Hole burning study of excited state structure and energy transfer dynamics of bacteriochlorophyll c in chlorosomes of green sulphur photosynthetic bacteria

Results of low temperature fluorescence and spectral hole burning experiments with whole cells and isolated chlorosomes of the green sulfur bacterium Chlorobium limicola containing BChl c are reported. At least two spectral forms of BChl c (short-wavelength and long-wavelength absorbing BChl c) were identified in the second derivative fluorescence spectra. The widths of persistent holes burned in the fluorescence spectrum of BChl c are determined by excited state lifetimes due to fast energy transfer. Different excited state lifetimes for both BChl c forms were observed. A site distribution function of the lowest excited state of chlorosomal BChl c was revealed. The excited state lifetimes are strongly influenced by redox conditions of the solution. At anaerobic conditions the lifetime of 5.3 ps corresponds to the rate of energy transfer between BChl c clusters. This time shortens to 2.6 ps at aerobic conditions. The shortening may be caused by introducing a quencher. Spectral bands observed in the fluorescence of isolated chlorosomes were attributed to monomeric and lower state aggregates of BChl c. These forms are not functionally connected with the chlorosome.Abbreviations BChl bacteriochlorophyll - EET electronic energy transfer - FWHM full width at half maximum - SDF site distribution function - RC reaction centre     

Ultrafast excited‐state dynamics in chlorosomes isolated from the photosynthetic filamentous green bacterium Chloroflexus aurantiacus

Bacteriochlorophyll (BChl) c pigments in the aggregated state are responsible for efficient light harvesting in chlorosomes of the filamentous anoxygenic photosynthetic bacterium, Chloroflexus (Cfx.) aurantiacus. Absorption of light creates excited states in the BChl c aggregates. After subpicosecond intrachlorosomal energy transfer, redistribution and relaxation, the excitation is transferred to the BChl a complexes and further to reaction centers on the picosecond time scale. In this work, the femtosecond excited state dynamics within BChl c oligomers of isolated Cfx. aurantiacus chlorosomes was studied by double difference pump‐probe spectroscopy at room temperature. Difference (A^light ? A^dark) spectra corresponding to excitation at 725 nm (blue side of the BChl c absorption band) were compared with those corresponding to excitation at 750 nm (red side of the BChl c absorption band). A very fast (time constant 70 ± 10 fs) rise kinetic component was found in the stimulated emission (SE) upon excitation at 725 nm. This component was absent at 750‐nm excitation. These data were explained by the dynamical red shift of the SE due to excited state relaxation. The nature and mechanisms of the ultrafast excited state dynamics in chlorosomal BChl c aggregates are discussed.     

The rate of Qx→Qy relaxation in bacteriochlorophylls of reaction centers from Rhodobacter sphaeroides determined by kinetics of the ultrafast carotenoid bandshift

Transient absorption changes induced by excitation of isolated reaction centers (RCs) from Rhodobacter sphaeroides with 600 nm laser pulses of 20 fs (full width at half maximum) were monitored in the wavelength region of 420–560 nm. The spectral features of the spectrum obtained are characteristic for an electrochromic band shift of the single carotenoid (Car) molecule spheroidene, which is an integral constituent of these RCs. This effect is assigned to an electrochromic bandshift of Car due to the local electric field of the dipole moment formed by electronic excitation of bacteriochlorophyll (BChl) molecule(s) in the neighborhood of Car. Based on the known distances between the pigments, the monomeric BChl (B_B) in the inactive B-branch is inferred to dominate this effect. The excitation of B_B at 600 nm leads to a transition into the S₂ state (Q_x band), which is followed by rapid internal conversion to the S₁ state (Q_y band), thus leading to a change of strength and orientation of the dipole moment, i.e., of the electric field acting on the Car molecule. Therefore, the time course of the electrochromic bandshift reflects the rate of the internal conversion from S₂ to S₁ of B_B. The evaluation of the kinetics leads to a value of 30 fs for this relaxation process. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Purification of the photosynthetic reaction center from <Emphasis Type="Italic">Heliobacterium modesticaldum</Emphasis>

We have developed a purification protocol for photoactive reaction centers (HbRC) from Heliobacterium modesticaldum. HbRCs were purified from solubilized membranes in two sequential chromatographic steps, resulting in the isolation of a fraction containing a single polypeptide, which was identified as PshA by LC–MS/MS of tryptic peptides. All polypeptides reported earlier as unknown proteins (in Heinnickel et al., Biochemistry 45:6756–6764, 2006; Romberger et al., Photosynth Res 104:293–303, 2010) are now identified by mass spectrometry to be the membrane-bound cytochrome c ₅₅₃ and four different ABC-type transporters. The purified PshA homodimer binds the following pigments: 20 bacteriochlorophyll (BChl) g, two BChl g′, two 8¹-OH-Chl a _F, and one 4,4′-diaponeurosporene. It lacks the PshB polypeptide binding the F_A and F_B [4Fe–4S] clusters. It is active in charge separation and exhibits a trapping time of 23 ps, as judged by time-resolved fluorescence studies. The charge recombination rate of the P₈₀₀ ⁺F_X⁻ state is 10–15 ms, as seen before. The purified HbRC core was able to reduce cyanobacterial flavodoxin in the light, exhibiting a K _M of 10 μM and a k _cat of 9.5 s⁻¹ under near-saturating light. There are ~1.6 menaquinones per HbRC in the purified complex. Illumination of frozen HbRC in the presence of dithionite can cause creation of a radical at g = 2.0046, but this is not a semiquinone. Furthermore, we show that high-purity HbRCs are very stable in anoxic conditions and even remain active in the presence of oxygen under low light.     

Probing binding site of bacteriochlorophyll a and carotenoid in the reconstituted LH1 complex from Rhodospirillum rubrum S1 by Stark spectroscopy

Stark spectroscopy is a powerful technique to investigate the electrostatic interactions between pigments as well as between the pigments and the proteins in photosynthetic pigment–protein complexes. In this study, Stark spectroscopy has been used to determine two nonlinear optical parameters (polarizability change Tr(Δα) and static dipole-moment change |Δμ| upon photoexcitation) of isolated and of reconstituted LH1 complexes from the purple photosynthetic bacterium, Rhodospirillum (Rs.) rubrum. The integral LH1 complex was prepared from Rs. rubrum S1, while the reconstituted complex was assembled by addition of purified carotenoid (all-trans-spirilloxanthin) to the monomeric subunit of LH1 from Rs. rubrum S1. The reconstituted LH1 complex has its Q_y absorption maximum at 878 nm. This is shifted to the blue by 3 nm in comparison to the isolated LH1 complex. The energy transfer efficiency from carotenoid to bacteriochlorophyll a (BChl a), which was determined by fluorescence excitation spectroscopy of the reconstituted LH1 complex, is increased to 40%, while the efficiency in the isolated LH1 complex is only 28%. Based on the differences in the values of Tr(Δα) and |Δμ|, between these two preparations, we can calculate the change in the electric field around the BChl a molecules in the two situations to be E _Δ ≈ 3.4 × 10⁵ [V/cm]. This change can explain the 3 nm wavelength shift of the Q_y absorption band in the reconstituted LH1 complex.     

Structural and spectropotentiometric analysis of Blastochloris viridis heterodimer mutant reaction center

Heterodimer mutant reaction centers (RCs) of Blastochloris viridis were crystallized using microfluidic technology. In this mutant, a leucine residue replaced the histidine residue which had acted as a fifth ligand to the bacteriochlorophyll (BChl) of the primary electron donor dimer M site (HisM200). With the loss of the histidine-coordinated Mg, one bacteriochlorophyll of the special pair was converted into a bacteriopheophytin (BPhe), and the primary donor became a heterodimer supermolecule. The crystals had dimensions 400 × 100 × 100 μm, belonged to space group P4₃2₁2, and were isomorphous to the ones reported earlier for the wild type (WT) strain. The structure was solved to a 2.5 Å resolution limit. Electron-density maps confirmed the replacement of the histidine residue and the absence of Mg. Structural changes in the heterodimer mutant RC relative to the WT included the absence of the water molecule that is typically positioned between the M side of the primary donor and the accessory BChl, a slight shift in the position of amino acids surrounding the site of the mutation, and the rotation of the M194 phenylalanine. The cytochrome subunit was anchored similarly as in the WT and had no detectable changes in its overall position. The highly conserved tyrosine L162, located between the primary donor and the highest potential heme C₃₈₀, revealed only a minor deviation of its hydroxyl group. Concomitantly to modification of the BChl molecule, the redox potential of the heterodimer primary donor increased relative to that of the WT organism (772 mV vs. 517 mV). The availability of this heterodimer mutant and its crystal structure provides opportunities for investigating changes in light-induced electron transfer that reflect differences in redox cascades.     

Glutathione peroxidase3 of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> protects phospholipids during cadmium-induced oxidative stress

The present study was undertaken to determine the role of glutathione peroxidase3 (gpx3) in phospholipid protection in cells. Wild-type (WT) cells showed an overall increase in phospholipids upon 50 μM cadmium (Cd)-treatment, whereas an untreated gpx3Δ strain showed a drastic reduction in overall phospholipids which was further reduced with 50 μM Cd. In WT cells, Cd-exposure increased the short chain fatty acids and decreased the unsaturated fatty acids and the magnitude was high in Cd-treated gpx3Δ cells. Purified recombinant gpx3p showed higher activity with phospholipid hydroperoxides than shorter hydroperoxides. An increase in gpx activity was observed in Cd-treated WT cells and no such alteration was observed in gpx3Δ. WT cells treated with Cd showed an increase in MDA over untreated, while untreated gpx3Δ cells themselves showed a higher level of MDA which was further enhanced with Cd-treatment. Iron, zinc and calcium levels were significantly altered in WT and gpx3Δ cells during Cd-treatment.     

Pathways of energy transformation in antenna reaction center complexes of Heliobacillus mobilis

The conversion of excitation energy in the antenna reaction center complex of Heliobacillus mobilis was investigated at 10 K as well as at 275 K by means of time-resolved absorbance difference spectroscopy of isolated membranes in the (sub)picosecond time range. Selective excitation of the primary electron acceptor, chlorophyll (Chl) a 670, and of the different spectral pools of bacteriochlorophyll (BChl) g (BChl g 778, BChl g 793, and BChl g 808) was applied. At 10 K, excitation at 770 or 793 nm resulted on the one hand in rapid energy transfer to BChl g 808 and on the other hand in fast charge separation from excited BChl g 793 ( approximately 1 ps). Once the excitations were on BChl g 808, the bleaching band shifted gradually to the red, from 806 to 813 nm, and charge separation from excited BChl g 808 occurred by a very slow process ( approximately 500 ps). The main purpose of our experiments was to answer the question whether an "alternative" pathway for charge separation exists upon excitation of Chl a 670. Our measurements showed that the amount of oxidized primary donor (P798(+)) relative to that of excited BChl g produced by excitation of Chl a 670 was considerably larger than upon direct excitation of BChl g. This indicates the existence of an alternative pathway for charge separation that does not involve excited antenna BChl g. This effect occurred at 10 K as well as at 275 K. The mechanism for this process is discussed in relation to different trapping models; it is concluded that charge separation occurs directly from excited Chl a 670.     

Concentration-Dependent Effects on Intracellular and Surface pH of Exposing <Emphasis Type="Italic">Xenopus</Emphasis> oocytes to Solutions Containing NH<Subscript>3</Subscript>/NH<Subscript>4</Subscript><Superscript>+</Superscript>

Others have shown that exposing oocytes to high levels of (10–20 mM) causes a paradoxical fall in intracellular pH (pH_i), whereas low levels (e.g., 0.5 mM) cause little pH_i change. Here we monitored pH_i and extracellular surface pH (pH_S) while exposing oocytes to 5 or 0.5 mM NH₃/NH₄ ⁺. We confirm that 5 mM causes a paradoxical pH_i fall (−ΔpH_i ≅ 0.2), but also observe an abrupt pH_S fall (−ΔpH_S ≅ 0.2)—indicative of NH₃ influx—followed by a slow decay. Reducing [NH₃/NH₄ ⁺] to 0.5 mM minimizes pH_i changes but maintains pH_S changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates −ΔpH_S at both levels. During removal of 0.5 or 5 mM NH₃/NH₄ ⁺, failure of pH_S to markedly overshoot bulk extracellular pH implies little NH₃ efflux and, thus, little free cytosolic NH₃/NH₄ ⁺. A new analysis of the effects of NH₃ vs. NH₄ ⁺ fluxes on pH_S and pH_i indicates that (a) NH₃ rather than NH₄ ⁺ fluxes dominate pH_i and pH_S changes and (b) oocytes dispose of most incoming NH₃. NMR studies of oocytes exposed to ¹⁵N-labeled show no significant formation of glutamine but substantial accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pH_i and pH_S demonstrate that NH₃ flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane—AmtB—enhances the flux of a gas across a biological membrane.

Oxidation state changes of the Mn<Subscript>4</Subscript>Ca cluster in Photosystem II

A detailed electronic structure of the Mn₄Ca cluster is required before two key questions for understanding the mechanism of photosynthetic water oxidation can be addressed. They are whether all four oxidizing equivalents necessary to oxidize water to O₂ accumulate on the four Mn ions of the oxygen-evolving complex, or do some ligand-centered oxidations take place before the formation and release of O₂ during the S₃ → [S₄] → S₀ transition, and what are the oxidation state assignments for the Mn during S-state advancement. X-ray absorption and emission spectroscopy of Mn, including the newly introduced resonant inelastic X-ray scattering spectroscopy have been used to address these questions. The present state of understanding of the electronic structure and oxidation state changes of the Mn₄Ca cluster in all the S-states, particularly in the S₂ to S₃ transition, derived from these techniques is described in this review.