Structure of the alternative complex III in a supercomplex with cytochrome oxidase.

Alternative complex III (ACIII) is a key component of the respiratory and/or photosynthetic electron transport chains of many bacteria1-3. Like complex III (also known as the bc1 complex), ACIII catalyses the oxidation of membrane-bound quinol and the reduction of cytochrome c or an equivalent electron carrier. However, the two complexes have no structural similarity4-7. Although ACIII has eluded structural characterization, several of its subunits are known to be homologous to members of the complex iron-sulfur molybdoenzyme (CISM) superfamily 8 , including the proton pump polysulfide reductase9,10. We isolated the ACIII from Flavobacterium johnsoniae with native lipids using styrene maleic acid copolymer11-14, both as an independent enzyme and as a functional 1:1 supercomplex with an aa3-type cytochrome c oxidase (cyt aa3). We determined the structure of ACIII to 3.4 Å resolution by cryo-electron microscopy and constructed an atomic model for its six subunits. The structure, which contains a [3Fe-4S] cluster, a [4Fe-4S] cluster and six haem c units, shows that ACIII uses known elements from other electron transport complexes arranged in a previously unknown manner. Modelling of the cyt aa3 component of the supercomplex revealed that it is structurally modified to facilitate association with ACIII, illustrating the importance of the supercomplex in this electron transport chain. The structure also resolves two of the subunits of ACIII that are anchored to the lipid bilayer with N-terminal triacylated cysteine residues, an important post-translational modification found in numerous prokaryotic membrane proteins that has not previously been observed structurally in a lipid bilayer.

Cryo-EM structures of Escherichia coli cytochrome bo3 reveal bound phospholipids and ubiquinone-8 in a dynamic substrate binding site.

Two independent structures of the proton-pumping, respiratory cytochrome bo3 ubiquinol oxidase (cyt bo3 ) have been determined by cryogenic electron microscopy (cryo-EM) in styrene-maleic acid (SMA) copolymer nanodiscs and in membrane scaffold protein (MSP) nanodiscs to 2.55- and 2.19-Å resolution, respectively. The structures include the metal redox centers (heme b, heme o3 , and CuB), the redox-active cross-linked histidine-tyrosine cofactor, and the internal water molecules in the proton-conducting D channel. Each structure also contains one equivalent of ubiquinone-8 (UQ8) in the substrate binding site as well as several phospholipid molecules. The isoprene side chain of UQ8 is clamped within a hydrophobic groove in subunit I by transmembrane helix TM0, which is only present in quinol oxidases and not in the closely related cytochrome c oxidases. Both structures show carbonyl O1 of the UQ8 headgroup hydrogen bonded to D75I and R71I In both structures, residue H98I occupies two conformations. In conformation 1, H98I forms a hydrogen bond with carbonyl O4 of the UQ8 headgroup, but in conformation 2, the imidazole side chain of H98I has flipped to form a hydrogen bond with E14I at the N-terminal end of TM0. We propose that H98I dynamics facilitate proton transfer from ubiquinol to the periplasmic aqueous phase during oxidation of the substrate. Computational studies show that TM0 creates a channel, allowing access of water to the ubiquinol headgroup and to H98I.

WITHDRAWN: Characterization of the supercomplex formed by the alternative complex III and the terminal aa3 oxidase from Flavobacterium johnsoniae isolated in styrene:maleic acid copolymer nanodiscs.

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Location of the Substrate Binding Site of the Cytochrome bo3 Ubiquinol Oxidase from Escherichia coli.

Cytochrome bo3 is a respiratory proton-pumping oxygen reductase that is a member of the heme-copper superfamily that utilizes ubiquinol-8 (Q8H2) as a substrate. The current consensus model has Q8H2 oxidized at a low affinity site (QL), passing electrons to a tightly bound quinone cofactor at a high affinity site (QH site) that stabilizes the one-electron reduced ubisemiquinone, facilitating the transfer of electrons to the redox active metal centers where O2 is reduced to water. The current work shows that the Q8 bound to the QH site is more dynamic than previously thought. In addition, mutations of residues at the QH site that do not abolish activity have been re-examined and shown to have properties expected of mutations at the substrate binding site (QL): an increase in the KM of the substrate ubiquinol-1 (up to 4-fold) and an increase in the apparent Ki of the inhibitor HQNO (up to 8-fold). The data suggest that there is only one binding site for ubiquinol in cyt bo3 and that site corresponds to the QH site.

The semiquinone at the Qi site of the bc1 complex explored using HYSCORE spectroscopy and specific isotopic labeling of ubiquinone in Rhodobacter sphaeroides via (13)C methionine and construction of a methionine auxotroph.

Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe (13)C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group (13)C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the (13)C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of (13)C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9 ) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly.

The mechanism of ubihydroquinone oxidation at the Qo-site of the cytochrome bc1 complex.

1. Recent results suggest that the major flux is carried by a monomeric function, not by an intermonomer electron flow. 2. The bifurcated reaction at the Qo-site involves sequential partial processes, - a rate limiting first electron transfer generating a semiquinone (SQ) intermediate, and a rapid second electron transfer in which the SQ is oxidized by the low potential chain. 3. The rate constant for the first step in a strongly endergonic, proton-first-then-electron mechanism, is given by a Marcus-Brønsted treatment in which a rapid electron transfer is convoluted with a weak occupancy of the proton configuration needed for electron transfer. 4. A rapid second electron transfer pulls the overall reaction over. Mutation of Glu-295 of cyt b shows it to be a key player. 5. In more crippled mutants, electron transfer is severely inhibited and the bell-shaped pH dependence of wildtype is replaced by a dependence on a single pK at ~8.5 favoring electron transfer. Loss of a pK ~6.5 is explained by a change in the rate limiting step from the first to the second electron transfer; the pK ~8.5 may reflect dissociation of QH. 6. A rate constant (<10(3)s(-1)) for oxidation of SQ in the distal domain by heme bL has been determined, which precludes mechanisms for normal flux in which SQ is constrained there. 7. Glu-295 catalyzes proton exit through H(+) transfer from QH, and rotational displacement to deliver the H(+) to exit channel(s). This opens a volume into which Q(-) can move closer to the heme to speed electron transfer. 8. A kinetic model accounts well for the observations, but leaves open the question of gating mechanisms. For the first step we suggest a molecular "escapement"; for the second a molecular ballet choreographed through coulombic interactions. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Inter-monomer electron transfer is too slow to compete with monomeric turnover in bc(1) complex.

The homodimeric bc(1) complexes are membrane proteins essential in respiration and photosynthesis. The ~11Å distance between the two b(L)-hemes of the dimer opens the possibility of electron transfer between them, but contradictory reports make such inter-monomer electron transfer controversial. We have constructed in Rhodobacter sphaeroides a heterodimeric expression system similar to those used before, in which the bc(1) complex can be mutated differentially in the two copies of cyt b to test for inter-monomer electron transfer, but found that genetic recombination by cross-over then occurs to produce wild-type homodimer. Selection pressure under photosynthetic growth always favored the homodimer over heterodimeric variants enforcing inter-monomer electron transfer, showing that the latter are not competitive. These results, together with kinetic analysis of myxothiazol titrations, demonstrate that inter-monomer electron transfer does not occur at rates competitive with monomeric turnover. We examine the results from other groups interpreted as demonstrating rapid inter-monomer electron transfer, conclude that similar mechanisms are likely to be in play, and suggest that such claims might need to be re-examined.

Real-time realizable mobile imaging photoplethysmography.

Photoplethysmography imaging (PPGI) sensors have attracted a significant amount of attention as they enable the remote monitoring of heart rates (HRs) and thus do not require any additional devices to be worn on fingers or wrists. In this study, we mounted PPGI sensors on a robot for active and autonomous HR (R-AAH) estimation. We proposed an algorithm that provides accurate HR estimation, which can be performed in real time using vision and robot manipulation algorithms. By simplifying the extraction of facial skin images using saturation (S) values in the HSV color space, and selecting pixels based on the most frequent S value within the face image, we achieved a reliable HR assessment. The results of the proposed algorithm using the R-AAH method were evaluated by rigorous comparison with the results of existing algorithms on the UBFC-RPPG dataset (n = 42). The proposed algorithm yielded an average absolute error (AAE) of 0.71 beats per minute (bpm). The developed algorithm is simple, with a processing time of less than 1 s (275 ms for an 8-s window). The algorithm was further validated on our own dataset (BAMI-RPPG dataset [n = 14]) with an AAE of 0.82 bpm.

Ligand binding and conformational dynamics of the E. coli nicotinamide nucleotide transhydrogenase revealed by hydrogen/deuterium exchange mass spectrometry.

Nicotinamide nucleotide transhydrogenases are integral membrane proteins that utilizes the proton motive force to reduce NADP+ to NADPH while converting NADH to NAD+. Atomic structures of various transhydrogenases in different ligand-bound states have become available, and it is clear that the molecular mechanism involves major conformational changes. Here we utilized hydrogen/deuterium exchange mass spectrometry (HDX-MS) to map ligand binding sites and analyzed the structural dynamics of E. coli transhydrogenase. We found different allosteric effects on the protein depending on the bound ligand (NAD+, NADH, NADP+, NADPH). The binding of either NADP+ or NADPH to domain III had pronounced effects on the transmembrane helices comprising the proton-conducting channel in domain II. We also made use of cyclic ion mobility separation mass spectrometry (cyclic IMS-MS) to maximize coverage and sensitivity in the transmembrane domain, showing for the first time that this technique can be used for HDX-MS studies. Using cyclic IMS-MS, we increased sequence coverage from 68 % to 73 % in the transmembrane segments. Taken together, our results provide important new insights into the transhydrogenase reaction cycle and demonstrate the benefit of this new technique for HDX-MS to study ligand binding and conformational dynamics in membrane proteins.

Energy transfer between the nicotinamide nucleotide transhydrogenase and ATP synthase of Escherichia coli.

Membrane bound nicotinamide nucleotide transhydrogenase (TH) catalyses the hydride transfer from NADH to NADP+. Under physiological conditions, this reaction is endergonic and must be energized by the pmf, coupled to transmembrane proton transport. Recent structures of transhydrogenase holoenzymes suggest new mechanistic details, how the long-distance coupling between hydride transfer in the peripheral nucleotide binding sites and the membrane-localized proton transfer occurs that now must be tested experimentally. Here, we provide protocols for the efficient expression and purification of the Escherichia coli transhydrogenase and its reconstitution into liposomes, alone or together with the Escherichia coli F1F0 ATP synthase. We show that E. coli transhydrogenase is a reversible enzyme that can also work as a NADPH-driven proton pump. In liposomes containing both enzymes, NADPH driven H+-transport by TH is sufficient to instantly fuel ATP synthesis, which adds TH to the pool of pmf generating enzymes. If the same liposomes are energized with ATP, NADPH production by TH is stimulated > sixfold both by a pH gradient or a membrane potential. The presented protocols and results reinforce the tight coupling between hydride transfer in the peripheral nucleotide binding sites and transmembrane proton transport and provide powerful tools to investigate their coupling mechanism.