The Oligomeric Subunit c Rotor in the Fo Sector of ATP Synthase: Unresolved Questions in Our Understanding of Function

We have proposed a model for the oligomeric c-rotor of the F_o sector of ATP synthase and its interaction with subunit a during H⁺-transport driven rotation. The model is based upon the solution structure of monomeric subunit c, determined by NMR, and an extensive series of cross-linking distance constraints between c subunits and between subunits c and a. To explain the complete set of cross-linking data, we have suggested that the second transmembrane helix rotates during its interaction with subunit a in the course of the H⁺-translocation cycle. The H⁺-transport coupled rotation of this helix is proposed to drive the stepwise movement of the c-oligomeric rotor. The model is testable and provides a useful framework for addressing questions raised by other experiments.     

Cytosolic pyruvate kinase: subunit composition,activity, and amount in developing castor and soybean seeds,and biochemical characterization of the purified castor seed enzyme

Antibodies against Brassica napus cytosolic pyruvate kinase (PK_c) (EC 2.7.1.40) were employed to examine PK_c subunit composition and developmental profiles in castor and soybean seeds. A 56-kDa immunoreactive polypeptide was uniformly detected on immunoblots of clarified extracts from developing castor endosperm or soybean embryos. Maximal PK_c activities occurred early in castor oil seed (COS) and soybean development (7.1 and 5.5 (μmol of pyruvate produced/min) g⁻¹ FW, respectively) and were up to 25-fold greater than those of fully mature seeds. Time-course studies revealed a close correlation between extractable PK_c activity and the relative amount of the immunoreactive 56-kDa PK_c polypeptide. PK_c from developing COS was purified 1,874-fold to homogeneity and a final specific activity of 73.1 (μmol of pyruvate produced/min) mg⁻¹ protein. Gel filtration and SDS-PAGE indicated that this PK_c exists as a 230-kDa homotetramer composed of 56-kDa subunits. The mass fingerprint of tryptic peptides of the 56-kDa COS PK_c subunit best matched three putative PK_cs from Arabidopsis thaliana. The purified enzyme was relatively heat-stable and displayed a broad pH optimum of 6.4. However, more efficient substrate utilization (in terms of V _max /K _m for phosphoenolpyruvate or ADP) was observed at pH 7.4. Glutamate was the most effective inhibitor, whereas aspartate functioned as an activator by partially relieving glutamate inhibition. Together with our previous studies, the results: (1) allow a model to be formulated regarding the coordinate allosteric control of PKc and phosphoenolpyruvate carboxylase by aspartate and glutamate in developing COS, and (2) provide further biochemical evidence that castor plant PK_c exists as tissue-specific isozymes that exhibit substantial differences in their respective physical and regulatory properties.     

ATP Synthases With Novel Rotor Subunits: New Insights into Structure,Function and Evolution of ATPases

ATPases with unusual membrane-embedded rotor subunits were found in both F₁F₀ and A₁A₀ ATP synthases. The rotor subunit c of A₁A₀ ATPases is, in most cases, similar to subunit c from F₀. Surprisingly, multiplied c subunits with four, six, or even 26 transmembrane spans have been found in some archaea and these multiplication events were sometimes accompanied by loss of the ion-translocating group. Nevertheless, these enzymes are still active as ATP synthases. A duplicated c subunit with only one ion-translocating group was found along with “normal” F₀ c subunits in the Na⁺ F₁F₀ ATP synthase of the bacterium Acetobacterium woodii. These extraordinary features and exceptional structural and functional variability in the rotor of ATP synthases may have arisen as an adaptation to different cellular needs and the extreme physicochemical conditions in the early history of life.     

Structural model of the transmembrane Fo rotary sector of H-transporting ATP synthase derived by solution NMR and intersubunit cross-linking in situ

H⁺-transporting, F₁F_o-type ATP synthases utilize a transmembrane H⁺ potential to drive ATP formation by a rotary catalytic mechanism. ATP is formed in alternating β subunits of the extramembranous F₁ sector of the enzyme, synthesis being driven by rotation of the γ subunit in the center of the F₁ molecule between the alternating catalytic sites . The H⁺ electrochemical potential is thought to drive γ subunit rotation by first coupling H⁺ transport to rotation of an oligomeric rotor of c subunits within the transmembrane F_o sector. The γ subunit is forced to turn with the c-oligomeric rotor due to connections between subunit c and the γ and ε subunits of F₁. In this essay we will review recent studies on the Escherichia coli F_o sector. The monomeric structure of subunit c, determined by NMR, shows that subunit c folds in a helical hairpin with the proton carrying Asp⁶¹ centered in the second transmembrane helix (TMH). A model for the structural organization of the c₁₀ oligomer in F_o was deduced from extensive cross-linking studies and by molecular modeling. The model indicates that the H⁺-carrying carboxyl of subunit c is occluded between neighboring subunits of the c₁₀ oligomer and that two c subunits pack in a “front-to-back” manner to form the H⁺ (cation) binding site. In order for protons to gain access to Asp⁶¹ during the protonation/deprotonation cycle, we propose that the outer, Asp⁶¹-bearing TMH-2s of the c-ring and TMHs from subunits composing the inlet and outlet channels must turn relative to each other, and that the swiveling motion associated with Asp⁶¹ protonation/deprotonation drives the rotation of the c-ring. The NMR structures of wild-type subunit c differs according to the protonation state of Asp⁶¹. The idea that the conformational state of subunit c changes during the catalytic cycle is supported by the cross-linking evidence in situ, and two recent NMR structures of functional mutant proteins in which critical residues have been switched between TMH-1 and TMH-2. The structural information is considered in the context of the possible mechanism of rotary movement of the c₁₀ oligomer during coupled synthesis of ATP.     

Two-Fold Symmetry of Crystalline DNA-ECORI Endonuclease Recognition Complexes

Abstract

Recognition complexes between EcoRI endonuclease and either of two synthetic oligonucleotides (sequences CGCGAATTCGCG and TCGCGAATTCGCG) crystallize in Space Group P321 with unit cell parameters a = 128 and c = 47 A and a = 118.4 and c = 49.7 A, respectively. Native diffraction data to 3 A resolution have been collected from the form containing the tridecameric sequence. Electrophoretic analyses of dissolved crystals demonstrate that this form contains DNA and protein in a ratio of one double helix per enzyme dimer. The most likely asymmetric unit contents are one 31,000 dalton enzyme subunit and one strand of DNA, yielding V_M values of 3.1 A³/dal and 2.8 A³/dal for the forms containing dodecameric and tridecameric DNA, respectively. This implies that the DNA-protein complex possesses two-fold rotational symmetry, which has been incorporated in the crystalline lattice.     

Potential role of subunit c of F0F1-ATPase and subunit c of storage body in the mitochondrial permeability transition. Effect of the phosphorylation status of subunit c on pore opening

Phosphorylated and non-phosphorylated forms of the F₀F₁-ATPase subunit c from rat liver mitochondria (RLM) were purified and their effect on the opening of the permeability transition pore (mPTP) was investigated. Addition of dephosphorylated subunit c to RLM induced mitochondrial swelling, decreased the membrane potential and reduced the Ca²⁺ uptake capacity, which was prevented by cyclosporin A. The same effect was observed in the presence of storage subunit c purified from livers of sheep affected with ceroid lipofuscinosis. In black-lipid bilayer membranes subunit c increased the conductance due to formation of single channels with fast and slow kinetics. The dephosphorylated subunit c formed channels with slow kinetics, i.e. the open state being of significantly longer duration than in the case of channels formed by the phosphorylated form that had short life spans and fast kinetics. The channels formed were cation-selective more so with the phosphorylated form. Subunit c of rat liver mitochondria was able to bind Ca²⁺. Collectively, the data allowed us to suppose that subunit c F₀F₁-ATPase might be a structural/regulatory component of mPTP exerting its role in dependence on phosphorylation status.     

Half channels mediating H transport and the mechanism of gating in the Fo sector of Escherichia coli F1Fo ATP synthase

H⁺-transporting F₁F_o ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F_o and F₁. H⁺ transport at the subunit a–c interface in trans-membranous F_o drives rotation of the c-ring within the membrane, with subunit c being bound in a complex with the γ and ε subunits extending from the membrane. Finally, the rotation of subunit γ within the α₃β₃ sector of F₁ mechanically drives ATP synthesis within the catalytic sites. In this review, we propose and provide evidence supporting the route of proton transfer via half channels from one side of the membrane to the other, and the mechanism of gating H⁺ binding to and release from Asp61 of subunit c, via conformational movements of Arg210 in subunit a. We propose that protons are gated from the inside of a four-helix bundle at the periplasmic side of subunit a to drive protonation of cAsp61, and that this gating movement is facilitated by the swiveling of trans-membrane helices (TMHs) 4 and 5 at the site of interaction with cAsp61 on the periphery of the c-ring. Proton release to the cytoplasmic half channel is facilitated by the movement of aArg210 as a consequence of this proposed helical swiveling. Finally, release from the cytoplasmic half channel is mediated by residues in a complex of interacting extra-membraneous loops formed between TMHs of both subunits a and c. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

A novel membrane-bound flavocytochrome c sulfide dehydrogenase from the colourless sulfur bacterium Thiobacillus sp. W5

A novel membrane-bound sulfide-oxidizing enzyme was purified 102-fold from the neutrophilic, obligately chemolithoautotrophic Thiobacillus sp. W5 by means of a six-step procedure. Spectral analysis revealed that the enzyme contains haem c and flavin. SDS-PAGE showed the presence of two types of subunit with molecular masses of 40 and 11 kDa. The smaller subunit contains covalently bound haem c, as was shown by haem staining. A combination of spectral analysis and the pyridine haemochrome test indicated that the sulfide-oxidizing heterodimer contains one molecule of haem c and one molecule of flavin. It appeared that the sulfide-oxidizing enzyme is a member of a small class of redox proteins, the flavocytochromes c, and is structurally most related to the flavocytochrome c sulfide dehydrogenase of the green sulfur bacterium Chlorobium limicola. The pH optimum of the enzyme is 8.6. At pH 9, the V _max was 2.1 ± 0.1 μmol cytochrome c (mg protein)^–1 min^–1, and the K _m values for sulfide and cytochrome c were 1.7 ± 0.4 μM and 3.8 ± 0.8 μM, respectively. Cyanide inhibited the enzyme by the formation of an N-5 adduct with the flavin moiety of the protein. On the basis of electron transfer stoichiometry, it seems likely that sulfur is the oxidation product. Received: 15 October 1996 / Accepted: 7 January 1997     

Kinetics of reextraction of Penicillin G from the organic phase after reactive extraction

The kinetics of reextraction of penicillin from the LA-2-penicillin complex-n-butylacetate solution by phosphate buffer was investigated experimentally and theoretically. The concentration time functions can be described by a mathematical model in a wide range of pH-values and complex concentrations.List of Symbols a _P [m²/m³] specific interfacial area - c _A , c _Ai [mMol] concentration of amine in the bulk of the organic phase and at the interface - c _AHP , c _AHPi [mMol] concentration of the complex in bulk of the organic phase and at the interface - c _H , c _Hi [mMol] concentration of the proton in the bulk of the aqueous phase and at the interface - c _P , c _Pi [mMol] concentration of the penicillin acid anion in the bulk of the aqueous phase and at the interface - J _A , J _AHP , J _P [mMol · cm · s^–1] fluxes of amine, complex and penicillin acid anion - k _A , k _AHP , k _P [cm · s^–1] mass transfer coefficients of amine, complex and penicillin acid anion - K _G [mMol²] equilibrium constant - t [k] extraction time     

ATP synthases: bioinformatic based insights into how their electrochemically driven motor comprised of subunits a and c might serve as a drug target

ATP synthases, widely distributed in bacteria, eukaryotic mitochondria and chloroplasts, are highly conserved multi-subunit complexes. Although the conserved acidic residue in the transmembrane helix of the c subunit functions in H⁺ transport, the surrounding residues differ among species. Such divergence could lead to different regulatory modes since pH-dependent H⁺ transport has been demonstrated in E. coli with a c subunit carrying an additional acidic residue in the helix. There is further divergence in the number of c subunits that form the ring structure which is determined by the higher ordered structure. Recently, it was suggested that certain chemicals recognize the a and c subunits of pathogenic bacterial F₀. Since there may be structural divergence even in well-conserved ATP synthases, the c subunit-ring as well as the a subunit in F₀ could be targets for drugs for specific bacterial species.     

The role of the membrane bound cytochromes of b- and c-type in the electron transport chain of Rhodobacter capsulatus

(1) The electron transport system of heterotrophically dark-grown Rhodobacter capsulatus was investigated using the wild-type strain MT1131 and the phototrophic non-competent (Ps^-) mutant MT-GS18 carrying deletions of the genes for cytochrome c ₁ and b of the bc ₁ complex and for cytochrome c ₂. (2) Spectroscopic and thermodynamic data demonstrate that deletion of both bc ₁ complex and cyt. c ₂ still leaves several haems of c- and b-type with Em_7.0 of +265 mV and +354 mV at 551–542 nm, and +415 mV and +275 mV at 561–575 nm, respectively. (3) Analysis of the oxidoreduction kinetic patterns of cytochromes indicated that cyt. b ₄₁₅ and cyt. b ₂₇₅ are reduced by either ascorbate-diaminodurene or NADH, respectively. (4) Growth on different carbon and nitrogen sources revealed that the membrane-bound electron transport chain of both MT1131 and MT-GS18 strains undergoes functional modifications in response to the composition of the growth medium used. (5) Excitation of membrane fragments from cells grown in malate minimal medium by a train of single turnover flashes of light led to a rapid oxidation of 32% of the membrane-bound c-type haem complement. Conversely, membranes prepared from peptone/yeast extract grown cells did not show cyt. c photooxidation. These results are discussed within the framework of an electron transport chain in which alternative pathways bypassing both the cyt. c ₂ and bc ₁ complex might involve high-potential membrane bound haems of b- and c-type.Abbreviations AA antimycin A - CCCP carbonylcyanide m-chlorophenyl hydrazone - CN^- cyanide - DAD diaminodurene - Q₂H₂ ubiquinol-2 - Q-pool ubiquinone-10 pool - RC photochemical reaction center     

A New Enzyme Species of p-Hydroxybenzoate Hydroxylase during Oxygenating Process Observed by the Stopped-flow Method

Sulfite ion (HSO₃ ^-) is one of the products when elemental sulfur is oxidized by the hydrogen sulfide:ferric ion oxidoreductase of Thiobacillus ferrooxidans AP19-3. Under the conditions in which HSO₃ ^- is accumulated in the cells, the iron oxidase of this bacterium was strongly inhibited by HSO₃ ^-. Since cytochrome c oxidase is one of the most important components of the iron oxidase enzyme system in T. ferrooxidans, effects of HSO₃ ^- on cytochrome c oxidase activity were studied with the plasma membranes of HSO₃ ^--resistant and -sensitive strains of T. ferrooxidans, OK1-50 and AP19-3. The enzyme activity of AP19-3 compared with OK1-50 was strongly inhibited by HSO₃ ^-. To investigate the inhibition mechanism of HSO₃ ^- in T. ferrooxidans, cytochrome c oxidases were purified from both strains to an electrophoretically homogeneous state. Cytochrome c oxidase activity of a purified OK1-50 enzyme was not inhibited by 5 mM HSO₃ ^-. In contrast, the same concentration of HSO₃ ^- inhibited the enzyme activity of AP19-3 50%, indicating that the cytochrome c oxidase of OK1-50 was more resistant to HSO₃ ^- than that of AP19-3. Cytochrome c oxidases purified from both strains were composed of three subunits. However, the molecular weight of the largest subunit differed between OK1-50 and AP19-3. Apparent molecular weights of the three subunits of cytochrome c oxidases were 53,000, 24,000, and 19,000 for strain AP19-3 and 55,000, 24,000, and 19,000 for strain OK1-50, respectively.     

A c Subunit with Four Transmembrane Helices and One Ion (Na+)-binding Site in an Archaeal ATP Synthase: IMPLICATIONS FOR c RING FUNCTION AND STRUCTURE*

The ion-driven membrane rotors of ATP synthases consist of multiple copies of subunit c, forming a closed ring. Subunit c typically comprises two transmembrane helices, and the c ring features an ion-binding site in between each pair of adjacent subunits. Here, we use experimental and computational methods to study the structure and specificity of an archaeal c subunit more akin to those of V-type ATPases, namely that from Pyrococcus furiosus. The c subunit was purified by chloroform/methanol extraction and determined to be 15.8 kDa with four predicted transmembrane helices. However, labeling with DCCD as well as Na⁺-DCCD competition experiments revealed only one binding site for DCCD and Na⁺, indicating that the mature c subunit of this A₁A_O ATP synthase is indeed of the V-type. A structural model generated computationally revealed one Na⁺-binding site within each of the c subunits, mediated by a conserved glutamate side chain alongside other coordinating groups. An intriguing second glutamate located in-between adjacent c subunits was ruled out as a functional Na⁺-binding site. Molecular dynamics simulations indicate that the c ring of P. furiosus is highly Na⁺-specific under in vivo conditions, comparable with the Na⁺-dependent V₁V_O ATPase from Enterococcus hirae. Interestingly, the same holds true for the c ring from the methanogenic archaeon Methanobrevibacter ruminantium, whose c subunits also feature a V-type architecture but carry two Na⁺-binding sites instead. These findings are discussed in light of their physiological relevance and with respect to the mode of ion coupling in A₁A_O ATP synthases.     

Structural studies of Bacillus subtilis glutamine synthetase. Further purification, sulfhydryl groups, and the NH2-terminal amino acid sequence.

A new procedure for the isolation of Bacillus subtilis glutamine synthetase in a high state of purity is described. Automated Edman degradation of the reduced and carboxy-methylated protein revealed a single NH₂-terminal amino acid sequence: H₂N-Ala-Lys- Tyr-Thr-Arg⁵-Glu-Asp-Ile-Gln-Lys¹⁰-Leu-Val-Ser-Glu-Ser¹⁵-CM-Cys-Val-Thr- Tyr-Ile²⁰-Ser-Leu-Gly-Phe-Ser²⁵-Asn-Ser-Leu-Gly- -. The recovery of phenylthiohydantoin(PTH)-amino acids and the single sequence obtained are consistent with the view that the dodecameric enzyme of molecular weight 600,000 is composed of identical subunits. Earlier observations of multiple sequences (80% PTH-Ala and 20% PTH-Gly as NH₂ terminal residues) appear to have been due to impurities removed by the final purification step described herein, which involves column chromatography on hydroxyapatite. Evidence for the existence of one disulfide bond and two free cysteine residues per subunit of dodecameric glutamine synthetase was obtained by alkylation of the denatured enzyme in the presence and absence of reducing agents. This distribution of the four cysteine residues in the enzyme monomer was confirmed by titration of the enzyme denatured in sodium dodecyl sulfate with 5,5′-dithiobis(2-nitrobenzoic acid).     

Electron-microscopic structure of the V-ATPase from mung bean

The vacuolar H⁺-ATPase from mung bean (Vigna radiata L. cv. Wilczek) was purified to homogeneity. The purified complex contained all the reported subunits from mung bean, but also included a 40-kDa subunit, corresponding to the membrane-associated subunit d, which has not previously been observed. The structure of the V-ATPase from mung bean was studied by electron microscopy of negatively stained samples. An analysis of over 6,000 single-particle images obtained by electron microscopy of the purified complex revealed that the complex, similar to other V-ATPases, is organized into two major domains V₁ and V_o with overall dimensions of 25 nm×13.7 nm and a stalk region connecting the V₁ and V_o domains. Several individual areas of protein density were observed in the stalk region, indicating its complexity. The projections clearly showed that the complex contained one central stalk and at least two peripheral stalks. Subcomplexes containing subunits A, B and E, dissociated from the tonoplast membrane by KI, were purified. The structure of the subcomplex was also studied by electron microscopy followed by single-molecule analysis of 13,000 projections. Our preliminary results reveal an area of high protein density at the bottom of the subcomplex immediately below the cavity formed by the A and B subunits, indicating the position of subunit E.Abbreviations MSA Multivariate statistical analysis - 2D, 3D Two-, three-dimensional - V-ATPase Vacuolar H⁺-ATPase     

Fluid Mechanical Matching of H-ATP Synthase Subunit c-Ring with Lipid Membranes Revealed by H Solid-State NMR

The F₁F_o-ATP synthase utilizes the transmembrane H⁺ gradient for the synthesis of ATP. F_o subunit c-ring plays a key role in transporting H⁺ through F_o in the membrane. We investigated the interactions of Escherichia coli subunit c with dimyristoylphosphatidylcholine (DMPC-d₅₄) at lipid/protein ratios of 50:1 and 20:1 by means of ²H-solid-state NMR. In the liquid-crystalline state of DMPC, the ²H-NMR moment values and the order parameter (S_CD) profile were little affected by the presence of subunit c, suggesting that the bilayer thickness in the liquid-crystalline state is matched to the transmembrane hydrophobic surface of subunit c. On the other hand, hydrophobic mismatch of subunit c with the lipid bilayer was observed in the gel state of DMPC. Moreover, the viscoelasticity represented by a square-law function of the ²H-NMR relaxation was also little influenced by subunit c in the fluid phase, in contrast with flexible nonionic detergents or rigid additives. Thus, the hydrophobic matching of the lipid bilayer to subunit c involves at least two factors, the hydrophobic length and the fluid mechanical property. These findings may be important for the torque generation in the rotary catalytic mechanism of the F₁F_o-ATPse molecular motor.     

Molecular Modeling of the Ternary Complex of Rusticyanin-Cytochrome c4-Cytochrome Oxidase: An Insight to Possible H-Bond Mediated Recognition and Electron Transfer Reaction in T.ferrooxidans

Abstract

The metabolism of Thiobacillus ferrooxidans involves electron transfer from the Fe⁺² ions in the extracellular environment to the terminal oxygen in the bacterial cytoplasm through a series of periplasmic proteins like Rusticyanin (RCy), Cytochrome (Cyt c₄), and Cytochrome oxidase (CcO). The energy minimization and MD studies reveal the stabilization of the three redox proteins in their ternary complex through the direct and water mediated H-bonds and electrostatic interaction. The surface exposed polar residues of the three proteins, i.e., RCy (His 143, Thr 146, Lys 81, Glu 20), Cyt c₄ (Asp 5, 15, 52, Ser 14, Glu 61), and CcO (Asp 135, Glu 126, 140, 142, Thr 177) formed the intermolecular hydrogen bonds and stabilized the ternary complex. The oxygen (O^εl) of Glu 126, 140, and 142 on subunit II of the CcO interact to the exposed side-chain and O^b atoms of the Asp 52 of Cyt c₄ and Glu 20 and Leu 12 of RCy. The Asp 135 of subunit II also forms H-bond with the N^ε atom of Lys 81 of RCy. The O^εl of Glu 61 of Cyt c₄ is also H-bonded to O^γ atom of Thr 177 of CcO. Solvation followed by MD studies of the ternary protein complex revealed the presence of seven water molecules in the interfacial region of the interacting proteins. Three of the seven water molecules (W 79, W 437, and W 606) bridged the three proteins by forming the hydrogen bonded network (with the distances ～ 2.10–2.95 Å) between the Lys 81 (RCy), Glu 61 (Cyt c₄), and Asp 135 (CcO). Another water molecule W 603 was H-bonded to Tyr 122 (CcO) and interconnected the Lys 81 (RCy) and Asp 135 (CcO) through the water molecules W 606 and W 437. The other two water molecules (W 21 and W 455) bridged the RCy to Cyt c₄ through H-bonds, whereas the remaining W 76 interconnected the His 53 (Cytc₄) to Glu 126 (CcO) with distances ～ 2.95–3.0 Å.     

Subunit Organization of the Stator Part of the F 0 Complex from Escherichia coli ATP Synthase

Membrane-bound ATP synthases (F₁F₀) catalyze the synthesis of ATP via a rotary catalyticmechanism utilizing the energy of an electrochemical ion gradient. The transmembrane potentialis supposed to propel rotation of a subunit c ring of F₀ together with subunits and of F₁,hereby forming the rotor part of the enzyme, whereas the remainder of the F₁F₀ complexfunctions as a stator for compensation of the torque generated during rotation. This reviewfocuses on our recent work on the stator part of the F₀ complex, e.g., subunits a and b. Usingepitope insertion and antibody binding, subunit a was shown to comprise six transmembranehelixes with both the N- and C-terminus oriented toward the cytoplasm. By use of circulardichroism (CD) spectroscopy, the secondary structure of subunit b incorporated intoproteoliposomes was determined to be 80% -helical together with 14% turn conformation, providingflexibility to the second stalk. Reconstituted subunit b together with isolated ac subcomplexwas shown to be active in proton translocation and functional F₁ binding revealing the nativeconformation of the polypeptide chain. Chemical crosslinking in everted membrane vesiclesled to the formation of subunit b homodimers around residues bQ37 to bL65, whereas bA32Ccould be crosslinked to subunit a, indicating a close proximity of subunits a and b near themembrane. Further evidence for the proposed direct interaction between subunits a and b wasobtained by purification of a stable ab ₂ subcomplex via affinity chromatography using Histags fused to subunit a or b. This ab ₂ subcomplex was shown to be active in proton translocationand F₁ binding, when coreconstituted with subunit c. Consequences of crosslink formationand subunit interaction within the F₁F₀ complex are discussed.     

Regulation of Energy Transduction and Electron Transfer in Cytochrome c Oxidase by Adenine Nucleotides

Cytochrome c oxidase from bovine heart contains seven high-affinity binding sites for ATP or ADP and three additional only for ADP. One binding site for ATP or ADP, located at the matrix-oriented domain of the heart-type subunit VIaH, increases the H⁺/e^– stoichiometry of the enzyme from heart or skeletal muscle from 0.5 to 1.0 when bound ATP is exchanged by ADP. Two further binding sites for ATP or ADP, located at the cytosolic and the matrix domain of subunit IV, increases the K _M for Cytochrome c and inhibit the respiratory activity at high ATP/ADP ratios, respectively. We propose that thermogenesis in mammals is related to subunit VIaL of cytochrome c oxidase with a H⁺/e^– stoichiometry of 0.5 compared to 1.0 in the enzyme from bacteria or ectotherm animals. This hypothesis is supported by the lack of subunit VIa isoforms in cytochrome c oxidase from fish.     

Cytochrome c peroxidase from Methylococcus capsulatus Bath

A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (E _m7 = –254 mV) and one low-spin, high-potential (E _m7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c ₅₅₅ as the electron donor, the K _M and V _max for peroxide reduction were 510 ± 100 nM and 425 ± 22 mol ferrocytochrome c ₅₅₅ oxidized min^–1 (mole cytochrome c peroxidase)^–1, respectively. Received: 6 January 1997 / Accepted: 27 May 1997