BBA - Bioenergetics (v.1857, #2)

ATP synthases (FoF1) are found ubiquitously in energy-transducing membranes of bacteria, mitochondria, and chloroplasts. These enzymes couple proton transport and ATP synthesis or hydrolysis through subunit rotation, which has been studied mainly by observing single molecules.In this review, we discuss the mechanism of rotational catalysis of ATP synthases, mainly that from Escherichia coli, emphasizing the high-speed and stochastic rotation including variable rates and an inhibited state. Single molecule studies combined with structural information of the bovine mitochondrial enzyme and mutational analysis have been informative as to an understanding of the catalytic site and the interaction between rotor and stator subunits. We discuss the similarity and difference in structure and inhibitory regulation of F1 from bovine and E. coli.Unlike the crystal structure of bovine F13β3γ), that of E. coli contains a ε subunit, which is a known inhibitor of bacterial and chloroplast F1 ATPases. The carboxyl terminal domain of E. coli ε (εCTD) interacts with the catalytic and rotor subunits (β and γ, respectively), and then inhibits rotation. The effects of phytopolyphenols on F1-ATPase are also discussed: one of them, piceatannol, lowered the rotational speed by affecting rotor/stator interactions.
Keywords: ATP synthase; F-ATPase; Rotational catalysis; Single molecule analysis; Phytopolyphenol; Epsilon subunit;

Identification of the coupling step in Na+-translocating NADH:quinone oxidoreductase from real-time kinetics of electron transfer by Nikolai P. Belevich; Yulia V. Bertsova; Marina L. Verkhovskaya; Alexander A. Baykov; Alexander V. Bogachev (141-149).
Bacterial Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) uses a unique set of prosthetic redox groups—two covalently bound FMN residues, a [2Fe–2S] cluster, FAD, riboflavin and a Cys4[Fe] center—to catalyze electron transfer from NADH to ubiquinone in a reaction coupled with Na+ translocation across the membrane. Here we used an ultra-fast microfluidic stopped-flow instrument to determine rate constants and the difference spectra for the six consecutive reaction steps of Vibrio harveyi Na+-NQR reduction by NADH. The instrument, with a dead time of 0.25 ms and optical path length of 1 cm allowed collection of visible spectra in 50-μs intervals. By comparing the spectra of reaction steps with the spectra of known redox transitions of individual enzyme cofactors, we were able to identify the chemical nature of most intermediates and the sequence of electron transfer events. A previously unknown spectral transition was detected and assigned to the Cys4[Fe] center reduction. Electron transfer from the [2Fe–2S] cluster to the Cys4[Fe] center and all subsequent steps were markedly accelerated when Na+ concentration was increased from 20 μM to 25 mM, suggesting coupling of the former step with tight Na+ binding to or occlusion by the enzyme. An alternating access mechanism was proposed to explain electron transfer between subunits NqrF and NqrC. According to the proposed mechanism, the Cys4[Fe] center is alternatively exposed to either side of the membrane, allowing the [2Fe–2S] cluster of NqrF and the FMN residue of NqrC to alternatively approach the Cys4[Fe] center from different sides of the membrane.
Keywords: Na+-translocating NADH:quinone oxidoreductase; Sodium transport; Electron transport; Redox reactions; Alternative access mechanism;

Optimizing multi-step B-side charge separation in photosynthetic reaction centers from Rhodobacter capsulatus by Kaitlyn M. Faries; Lucas L. Kressel; Nicholas P. Dylla; Marc J. Wander; Deborah K. Hanson; Dewey Holten; Philip D. Laible; Christine Kirmaier (150-159).
Using high-throughput methods for mutagenesis, protein isolation and charge-separation functionality, we have assayed 40 Rhodobacter capsulatus reaction center (RC) mutants for their P+  QB yield (P is a dimer of bacteriochlorophylls and Q is a ubiquinone) as produced using the normally inactive B-side cofactors BB and HB (where B is a bacteriochlorophyll and H is a bacteriopheophytin). Two sets of mutants explore all possible residues at M131 (M polypeptide, native residue Val near HB) in tandem with either a fixed His or a fixed Asn at L181 (L polypeptide, native residue Phe near BB). A third set of mutants explores all possible residues at L181 with a fixed Glu at M131 that can form a hydrogen bond to HB. For each set of mutants, the results of a rapid millisecond screening assay that probes the yield of P+  QB are compared among that set and to the other mutants reported here or previously. For a subset of eight mutants, the rate constants and yields of the individual B-side electron transfer processes are determined via transient absorption measurements spanning 100 fs to 50 μs. The resulting ranking of mutants for their yield of P+  QB from ultrafast experiments is in good agreement with that obtained from the millisecond screening assay, further validating the efficient, high-throughput screen for B-side transmembrane charge separation. Results from mutants that individually show progress toward optimization of P+  HB  → P+  QB electron transfer or initial P* → P+  HB conversion highlight unmet challenges of optimizing both processes simultaneously.Display Omitted
Keywords: Directionality; Asymmetry; Picosecond; Membrane; Saturation mutagenesis; Charge recombination;

Heme A is a prosthetic group unique for cytochrome a-type respiratory oxidases in mammals, plants and many microorganisms. The poorly understood integral membrane protein heme A synthase catalyzes the synthesis of heme A from heme O. In bacteria, but not in mitochondria, this enzyme contains one or two pairs of cysteine residues that are present in predicted hydrophilic polypeptide loops on the extracytoplasmic side of the membrane. We used heme A synthase from the eubacterium Bacillus subtilis and the hyperthermophilic archeon Aeropyrum pernix to investigate the functional role of these cysteine residues. Results with B. subtilis amino acid substituted proteins indicated the pair of cysteine residues in the loop connecting transmembrane segments I and II as being essential for catalysis but not required for binding of the enzyme substrate, heme O. Experiments with isolated A. pernix and B. subtilis heme A synthase demonstrated that a disulfide bond can form between the cysteine residues in the same loop and also between loops showing close proximity of the two loops in the folded enzyme protein.Based on the findings, we propose a classification scheme for the four discrete types of heme A synthase found so far in different organisms and propose that essential cysteinyls mediate transfer of reducing equivalents required for the oxygen-dependent catalysis of heme A synthesis from heme O.Display Omitted
Keywords: Heme biosynthesis; Cytochrome a; Bacillus subtilis; Aeropyrum pernix; CtaA; COX15; Protein disulfide;

The solution structure of the soluble form of the lipid-modified azurin from Neisseria gonorrhoeae, the electron donor of cytochrome c peroxidase by Cláudia S. Nóbrega; Ivo H. Saraiva; Cíntia Carreira; Bart Devreese; Manolis Matzapetakis; Sofia R. Pauleta (169-176).
Neisseria gonorrhoeae colonizes the genitourinary track, and in these environments, especially in the female host, the bacteria are subjected to low levels of oxygen, and reactive oxygen and nitrosyl species. Here, the biochemical characterization of N. gonorrhoeae Laz is presented, as well as, the solution structure of its soluble domain determined by NMR. N. gonorrhoeae Laz is a type 1 copper protein of the azurin-family based on its spectroscopic properties and structure, with a redox potential of 277 ± 5 mV, at pH 7.0, that behaves as a monomer in solution. The globular Laz soluble domain adopts the Greek-key motif, with the copper center located at one end of the β-barrel coordinated by Gly48, His49, Cys113, His118 and Met122, in a distorted trigonal geometry. The edge of the His118 imidazole ring is water exposed, in a surface that is proposed to be involved in the interaction with its redox partners. The heterologously expressed Laz was shown to be a competent electron donor to N. gonorrhoeae cytochrome c peroxidase. This is an evidence for its involvement in the mechanism of protection against hydrogen peroxide generated by neighboring lactobacilli in the host environment.Display Omitted
Keywords: Neisseria; Copper protein; Azurin; Laz; Cytochrome c peroxidase; Solution NMR structure;

The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase by Dhirendra Singh; Hendrik Sielaff; Lavanya Sundararaman; Shashi Bhushan; Gerhard Grüber (177-187).
A1AO ATP synthases couple ion-transport of the AO sector and ATP synthesis/hydrolysis of the A3B3-headpiece via their stalk subunits D and F. Here, we produced and purified stable A3B3D- and A3B3DF-complexes of the Methanosarcina mazei Gö1 A-ATP synthase as confirmed by electron microscopy. Enzymatic studies with these complexes showed that the M. mazei Gö1 A-ATP synthase subunit F is an ATPase activating subunit. The maximum ATP hydrolysis rates (V max ) of A3B3D and A3B3DF were determined by substrate-dependent ATP hydrolysis experiments resulting in a V max of 7.9 s− 1 and 30.4 s− 1, respectively, while the K M is the same for both. Deletions of the N- or C-termini of subunit F abolished the effect of ATP hydrolysis activation. We generated subunit F mutant proteins with single amino acid substitutions and demonstrated that the subunit F residues S84 and R88 are important in stimulating ATP hydrolysis. Hybrid formation of the A3B3D-complex with subunit F of the related eukaryotic V-ATPase of Saccharomyces cerevisiae or subunit ε of the F-ATP synthase from Mycobacterium tuberculosis showed that subunit F of the archaea and eukaryotic enzymes are important in ATP hydrolysis.
Keywords: Subunit F; A1AO ATP synthase; ATP synthase; Methanosarcina mazei Gö1; Archaea; Bioenergetics;