BBA - Bioenergetics (v.1807, #7)

A combined quantum chemical and crystallographic study on the oxidized binuclear center of cytochrome c oxidase by Ville R.I. Kaila; Esko Oksanen; Adrian Goldman; Dmitry A. Bloch; Michael I. Verkhovsky; Dage Sundholm; Mårten Wikström (769-778).
Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain. By reducing oxygen to water, it generates a proton gradient across the mitochondrial or bacterial membrane. Recently, two independent X-ray crystallographic studies ((Aoyama et al. Proc. Natl. Acad. Sci. USA 106 (2009) 2165–2169) and (Koepke et al. Biochim. Biophys. Acta 1787 (2009) 635–645)), suggested that a peroxide dianion might be bound to the active site of oxidized CcO. We have investigated this hypothesis by combining quantum chemical calculations with a re-refinement of the X-ray crystallographic data and optical spectroscopic measurements. Our data suggest that dianionic peroxide, superoxide, and dioxygen all form a similar superoxide species when inserted into a fully oxidized ferric/cupric binuclear site (BNC). We argue that stable peroxides are unlikely to be confined within the oxidized BNC since that would be expected to lead to bond splitting and formation of the catalytic P intermediate. Somewhat surprisingly, we find that binding of dioxygen to the oxidized binuclear site is weakly exergonic, and hence, the observed structure might have resulted from dioxygen itself or from superoxide generated from O2 by the X-ray beam. We show that the presence of O2 is consistent with the X-ray data. We also discuss how other structures, such as a mixture of the aqueous species (H2O + OH and H2O) and chloride fit the experimental data.► The oxidized active site of cytochrome c oxidase was studied by DFT. ► Results from DFT calculations were compared to X-ray diffraction data. ► Oxygenous species form a superoxide compound in the oxidized active site. ► Dianionic peroxide is expected to be cleaved in the oxidized active site. ► Dioxygen is consistent with the experimentally observed electron density.
Keywords: Heme-copper oxidases; Oxygen binding; Density functional theory (DFT); X-ray refinement;

Spectral components of the α-band of cytochrome oxidase by N. Kim; M.O. Ripple; R. Springett (779-787).
Oxidative redox titrations of the mitochondrial cytochromes were performed in near-anoxic RAW 264.7 cells by inhibiting complex I. Cytochrome oxidation changes were measured with multi-wavelength spectroscopy and the ambient redox potential was calculated from the oxidation state of endogenous cytochrome c. Two spectral components were separated in the α-band range of cytochrome oxidase and they were identified as the difference spectrum of heme a when it has a high (a H) or low (a L) midpoint potential (E m) by comparing their occupancy during redox titrations carried out when the membrane potential (ΔΨ) was dissipated with a protonophore to that predicted by the neoclassical model of redox cooperativity. The difference spectrum of a L has a maximum at 605 nm whereas the spectrum of a H has a maximum at 602 nm. The ΔΨ-dependent shift in the E m of a H was too great to be accounted for by electron transfer from cytochrome c to heme a against ΔΨ but was consistent with a model in which a H is formed after proton uptake against ΔΨ suggesting that the spectral changes are the result of protonation. A stochastic simulation was implemented to model oxidation states, proton uptake and E m changes during redox titrations. The redox anti-cooperativity between heme a and heme a 3, and proton binding, could be simulated with a model where the pump proton interacted with heme a and the substrate proton interacted with heme a 3 with anti-cooperativity between proton binding sites, but not with a single proton binding site coupled to both hemes.Display Omitted► Each heme has a protonation site that raises its midpoint potential. ► Protonation blue-shifts the α-band absorption spectrum of heme a. ► The spectral components of heme a can be decomposed with full spectral fitting. ► Redox cooperativity results from anticooperativity between the protons.
Keywords: Cytochrome oxidase; Redox titration; Midpoint potential; Optical spectroscopy; Proton pumping; Membrane potential;

The Q cycle of cytochrome bc complexes: A structure perspective by William A. Cramer; S. Saif Hasan; Eiki Yamashita (788-802).
Aspects of the crystal structures of the hetero-oligomeric cytochrome bc 1 and b 6 f (“bc”) complexes relevant to their electron/proton transfer function and the associated redox reactions of the lipophilic quinones are discussed. Differences between the b 6 f and bc 1 complexes are emphasized. The cytochrome bc 1 and b 6 f dimeric complexes diverge in structure from a core of subunits that coordinate redox groups consisting of two bis-histidine coordinated hemes, a heme b n and b p on the electrochemically negative (n) and positive (p) sides of the complex, the high potential [2Fe–2S] cluster and c-type heme at the p-side aqueous interface and aqueous phase, respectively, and quinone/quinol binding sites on the n- and p-sides of the complex. The bc 1 and b 6 f complexes diverge in subunit composition and structure away from this core. b 6 f Also contains additional prosthetic groups including a c-type heme c n on the n-side, and a chlorophyll a and β-carotene. Common structure aspects; functions of the symmetric dimer . (I) Quinone exchange with the bilayer. An inter-monomer protein-free cavity of approximately 30 Å along the membrane normal × 25 Å (central inter-monomer distance) × 15 Å (depth in the center), is common to both bc 1 and b 6 f complexes, providing a niche in which the lipophilic quinone/quinol (Q/QH2) can be exchanged with the membrane bilayer. (II) Electron transfer. The dimeric structure and the proximity of the two hemes b p on the electrochemically positive side of the complex in the two monomer units allow the possibility of two alternate routes of electron transfer across the complex from heme b p to b n: intra-monomer and inter-monomer involving electron cross-over between the two hemes b p. A structure-based summary of inter-heme distances in seven bc complexes, representing mitochondrial, chromatophore, cyanobacterial, and algal sources, indicates that, based on the distance parameter, the intra-monomer pathway would be favored kinetically. (III) Separation of quinone binding sites. A consequence of the dimer structure and the position of the Q/QH2 binding sites is that the p-side QH2 oxidation and n-side Q reduction sites are each well separated. Therefore, in the event of an overlap in residence time by QH2 or Q molecules at the two oxidation or reduction sites, their spatial separation would result in minimal steric interference between extended Q or QH2 isoprenoid chains. (IV) Trans-membrane QH 2 /Q transfer. (i) n/p-side QH2/Q transfer may be hindered by lipid acyl chains; (ii) the shorter less hindered inter-monomer pathway across the complex would not pass through the center of the cavity, as inferred from the n-side antimycin site on one monomer and the p-side stigmatellin site on the other residing on the same surface of the complex. (V) Narrow p-side portal for QH 2 /Q passage. The [2Fe–2S] cluster that serves as oxidant, and whose histidine ligand serves as a H+ acceptor in the oxidation of QH2, is connected to the inter-monomer cavity by a narrow extended portal, which is also occupied in the b 6 f complex by the 20 carbon phytyl chain of the bound chlorophyll.► Comparative discussion of crystal structures, biochemical properties, and functions of cytochrome bc 1 complexes and the b 6 f complex in oxygenic photosynthesis. ► Structure-function of lipids integral to the complex. ► Differences in Q cycle in bc 1 and b 6 f complexes. ► Steric problems in entry/exit of lipophilic quinol/quinone in p-side portal of cytochrome bc complexes. ► Implications of structure for intra- and inter-monomer pathways of electron transfer.
Keywords: Cytochrome bc 1/b 6 f complex; Electron transfer; Energy transduction; Plasto-/ubiquinone; Electrochemical potential;

The 6xHis-tag-pscA gene, which was genetically engineered to express N-terminally histidine (His)-tagged PscA, was inserted into a coding region of the recA gene in the green sulfur bacterium Chlorobaculum tepidum (C. tepidum). Although the inactivation of the recA gene strongly suppressed a homologous recombination in C. tepidum genomic DNA, the mutant grew well under normal photosynthetic conditions. The His-tagged reaction center (RC) complex could be obtained simply by Ni2+-affinity chromatography after detergent solubilization of chlorosome-containing membranes. The complex consisted of three subunits, PscA, PscB, and PscC, in addition to the Fenna–Matthews–Olson protein, but there was no PscD. Low-temperature EPR spectroscopic studies in combination with transient absorption measurements indicated that the complex contained all intrinsic electron transfer cofactors as detected in the wild-type strain. Furthermore, the LC/MS/MS analysis revealed that the core protein consisted of a mixture of a His-/His-tagged PscA homodimer and a non-/His-tagged PscA heterodimer. The development of the pscA gene duplication method presented here, thus, enables not only a quick and large-scale preparation of the RC complex from C. tepidum but also site-directed mutagenesis experiments on the artificially incorporated 6xHis-tag-pscA gene itself, since the expression of the authentic PscA/PscA homodimeric RC complex could complement any defect in mutated His-tagged PscA. This method would provide an invaluable tool for structural and functional analyses of the homodimeric type 1 RC complex.Display Omitted► Homodimeric type 1 RC in green sulfur bacteria consists of two identical PscA core subunits. ► Insertion of the His-pscA gene into the recA region. ► The His-tagged RC complex has no PscD subunit but shows intrinsic electron transfer activity. ► Evidence of the heterogeneous tag-attachment by LC/MS/MS analysis. ► A site-directed mutagenesis on the His-pscA gene would be possible.
Keywords: Green sulfur bacteria; Type 1 reaction center; Histidine tag; Electron paramagnetic resonance; LC/MS/MS; Iron–sulfur cluster;

Stabilization of the peroxy intermediate in the oxygen splitting reaction of cytochrome cbb 3 by Vivek Sharma; Mårten Wikström; Ville R.I. Kaila (813-818).
The proton-pumping cbb 3-type cytochrome c oxidases catalyze cell respiration in many pathogenic bacteria. For reasons not yet understood, the apparent dioxygen (O2) affinity in these enzymes is very high relative to other members of the heme-copper oxidase (HCO) superfamily. Based on density functional theory (DFT) calculations on intermediates of the oxygen scission reaction in active-site models of cbb 3- and aa 3-type oxidases, we find that a transient peroxy intermediate (I P , Fe[III]–OOH) is ~ 6 kcal/mol more stable in the former case, resulting in more efficient kinetic trapping of dioxygen and hence in a higher apparent oxygen affinity. The major molecular basis for this stabilization is a glutamate residue, polarizing the proximal histidine ligand of heme b 3 in the active site.Display Omitted► The high oxygen affinity of cbb 3-type oxidases is studied by DFT calculations. ► A stabilization of a peroxy intermediate is found relative to aa 3-type oxidases. ► A conserved glutamate polarizes the active site heme in the cbb 3-enzyme. ► An electrostatic perturbation result in an efficient kinetic trapping of dioxygen.
Keywords: Density Functional Theory (DFT); cbb 3-type cytochrome c oxidase; Oxygen activation; Oxygen affinity; Heme-copper oxidases;

Loss of dopamine (DA) homeostasis may be a contributing factor to cell damage in Parkinson's disease (PD). Past studies showing deleterious effects of DA on mitochondrial function, however, have been inconsistent raising questions about mitochondria as a downstream target for DA. Issues such as the dopamine species i.e., reduced or oxidized, time of exposure and the effect of major metabolites such as 3,4-dihydrophenylacetic acid (DOPAC) may contribute to the disparate findings. The present study used isolated, lysed rat brain mitochondria to characterize the effects of oxidized or reduced DA and DOPAC on complex activities of the electron transport chain (ETC). Time of exposure and quantitation of reduced or oxidized catachols for DA and DOPAC were monitored for all experiments. Reduced DA and DOPAC with or without a 30 min preincubation had no affect on NADH oxidase activity which monitors the activities of complexes I, III and IV. Complex II activity was inhibited by reduced DA (≥ 500 μM), but not by reduced DOPAC and was significantly attenuated by SOD suggesting reactive oxygen species involvement. In contrast, fully oxidized DA and DOPAC dose dependently inhibited NADH oxidase, complex I and complex III activities with IC50s in the 50–200 μM range. No preincubation was required for inhibition with the catechols when they were fully oxidized. Oxidized DA inhibited complex I only when exposure occurred during stimulated electron flow, suggesting covalent binding of quinones to proteins within active sites of the complex. In intact, well coupled mitochondria, extramitochondrial DA was shown to access the mitochondrial matrix in a dose, time and energy-dependent fashion. The findings suggest that many of the reported inconsistencies with regards to the effects of DA and DOPAC on ETC function can be attributed to the oxidized state of the catechol at the time of exposure. In addition, the findings provide possible downstream targets for DA that could contribute to the vulnerability of dopaminergic neurons in PD.► Oxidized dopamine and DOPAC inhibit mitochondrial complexes I and II with µM potencies. ► Reduced catechols inhibit only complex II with low potency. ► Exposure time in relation to catechol oxidation is important to inhibition. ► Extramitochondrial dopamine can access the mitochondrial matrix.
Keywords: Catechol; Neuron; Mitochondrion; Neurodegeneration; Parkinson's; Brain;

The electronic structures of the S 2 states of the oxygen-evolving complexes of photosystem II in plants and cyanobacteria in the presence and absence of methanol by Ji-Hu Su; Nicholas Cox; William Ames; Dimitrios A. Pantazis; Leonid Rapatskiy; Thomas Lohmiller; Leonid V. Kulik; Pierre Dorlet; A. William Rutherford; Frank Neese; Alain Boussac; Wolfgang Lubitz; Johannes Messinger (829-840).
The electronic properties of the Mn4O x Ca cluster in the S 2 state of the oxygen-evolving complex (OEC) were studied using X- and Q-band EPR and Q-band 55Mn-ENDOR using photosystem II preparations isolated from the thermophilic cyanobacterium T. elongatus and higher plants (spinach). The data presented here show that there is very little difference between the two species. Specifically it is shown that: (i) only small changes are seen in the fitted isotropic hyperfine values, suggesting that there is no significant difference in the overall spin distribution (electronic coupling scheme) between the two species; (ii) the inferred fine-structure tensor of the only MnIII ion in the cluster is of the same magnitude and geometry for both species types, suggesting that the MnIII ion has the same coordination sphere in both sample preparations; and (iii) the data from both species are consistent with only one structural model available in the literature, namely the Siegbahn structure [Siegbahn, P. E. M. Accounts Chem. Res. 2009, 42, 1871–1880, Pantazis, D. A. et al., Phys. Chem. Chem. Phys. 2009, 11, 6788–6798]. These measurements were made in the presence of methanol because it confers favorable magnetic relaxation properties to the cluster that facilitate pulse-EPR techniques. In the absence of methanol the separation of the ground state and the first excited state of the spin system is smaller. For cyanobacteria this effect is minor but in plant PS II it leads to a break-down of the S T  = ½ spin model of the S 2 state. This suggests that the methanol–OEC interaction is species dependent. It is proposed that the effect of small organic solvents on the electronic structure of the cluster is to change the coupling between the outer Mn (MnA) and the other three Mn ions that form the trimeric part of the cluster (MnB, MnC, MnD), by perturbing the linking bis-μ-oxo bridge. The flexibility of this bridging unit is discussed with regard to the mechanism of O-O bond formation.► The S 2 electronic structures of the OECs of T. elongatus and plant PS II are similar. ► Both can be explained using the same coupling scheme. ► The inferred on-site ZFS of the MnIII changes for the two OECs. ► Methanol changes the electronic coupling scheme.
Keywords: EPR; 55Mn-ENDOR; Photosystem II; OEC; Mn4O x Ca cluster; Methanol; Orbach process; Raman process; Spin Hamiltonian;

High light stress and the one-helix LHC-like proteins of the cryptophyte Guillardia theta by Christiane Funk; Meriem Alami; Tania Tibiletti; Beverley R. Green (841-846).
Cryptophytes like the cryptomonad Guillardia theta are part of the marine phytoplankton and therefore major players in global carbon and biogeochemical cycles. Despite the importance for the cell in being able to cope with large changes in illumination on a daily basis, very little is known about photoprotection mechanisms in cryptophytes. Here, we show that Guillardia theta is able to perform non-photochemical quenching, although none of the usual xanthophyll cycle pigments (e.g., zeaxanthin, diadinoxanthin, diatoxanthin) are present at detectable levels. Instead, acclimation to high light intensity seems to involve an increase of alloxanthin. Guillardia theta has genes for 2 one-helix “light-harvesting-like” proteins, related to some cyanobacterial genes which are induced in response to high light stress. Both the plastid-encoded gene (hlipP) and the nucleomorph-encoded gene (HlipNm) are expressed, but transcript levels decrease rather than increase during high light exposure, suggesting that they are not involved in a high light stress response. The HlipNm protein was detected with a specific antibody; expression was constant, independent of the light exposure.► The cryptomonad algae Guillardia theta is part of the marine phytoplankton. ► It performs non-photochemical quenching without the usual xanthophyll cycle pigments. ► G. theta contains two genes coding for one-helical light-harvesting-like proteins. ► Both the plastid- (hlipP) and the nucleomorph-encoded gene (HlipNm) are expressed. ► Surprisingly, expression of the hlips decreases during high light stress.
Keywords: Guillardia theta; Cryptomonad; High light inducible protein; Non-photochemical quenching; Light-harvesting-like protein;

We examined energy transfer dynamics from the photosystem II reaction center (PSII-RC) in intact red algae cells of Porphyridium cruentum, Bangia fuscopurpurea, Porphyra yezoensis, Chondrus giganteus, and Prionitis crispata. Time resolved fluorescence measurements were conducted in the range of 0–80 ns at − 196 °C. The delayed fluorescence spectra were then determined, where the delayed fluorescence was derived from the charge recombination between P680+ and pheophytin a in PSII-RC. Therefore, the delayed fluorescence spectrum reflected the energy migration processes including PSII-RC. All samples examined showed prominent distribution of delayed fluorescence in PSII and PSI, which suggests that a certain amount of PSII attaches to PSI to share excitation energy in red algae. The energy transfer from PSII to PSI was found to be dominant when the amount of phycoerythrobilin was increased.► Energy transfer from PSII to PSI (spillover) was monitored using spectroscopy. ► Time-resolved fluorescence spectra of intact cells of red algae were analyzed. ► Delayed fluorescence derived from charge recombination in PSII distributed to PSI. ► Spillover occurred frequently in red algae containing large amount of phycobilisome.
Keywords: Spillover; Red algae; Delayed fluorescence; Phycobilisome; Red chlorophyll;