Polypeptides protonation complexes

ATP synthase actually consists of two principal complexes. The spheres observed in electron micrographs make up the Fj unit, which catalyzes ATP synthesis. These Fj spheres are attached to an integral membrane protein aggregate called the Fq unit. Fj consists of five polypeptide chains named a, j3, y, 8, and e, with a subunit stoichiometry ajjSaySe (Table 21.3). Fq consists of three hydrophobic subunits denoted by a, b, and c, with an apparent stoichiometry of ajbgCg.ig- Fq forms the transmembrane pore or channel through which protons move to drive ATP synthesis. The a, j3, y, 8, and e subunits of Fj contain 510, 482, 272, 146, and 50 amino acids, respectively, with a total molecular mass... 

The ATP synthase (EC3.6.1.34, complex V) that transports H"" is a complex molecular machine. The enzyme consists of two parts—a proton channel (Fq, for oligomycin-sensitive ) that is integrated into the membrane and a catalytic unit (Fi) that protrudes into the matrix. The Fo part consists of 12 membrane-spanning c-peptides and one a-subunit. The head of the Fi part is composed of three a and three p subunits, between which there are three active centers. The stem between Fo and Fi consists of one y and one e subunit. Two more polypeptides, b and 8, form a kind of stator, fixing the a and p subunits relative to the Fo part. 

FIGURE 19-33 Bacterial respiratory chain, (a) Shown here are the respiratory carriers of the inner membrane of E. coli. Eubacteria contain a minimal form of Complex I, containing all the prosthetic groups normally associated with the mitochondrial complex but only 14 polypeptides. This plasma membrane complex transfers electrons from NADH to ubiquinone or to (b) menaquinone, the bacterial equivalent of ubiquinone, while pumping protons outward and creating an electrochemical potential that drives ATP synthesis. 

The enzyme is a hexamer, actually a dimer of trimers made up of 291-residue polypeptide chains.28 Aceto-acetyl-CoA is a competitive inhibitor which binds into the active site and locates it. From the X-ray structure of the enzyme-inhibitor complex it can be deduced that the carboxylate group of E144 abstracts a proton from a water molecule to provide the hydroxyl ion that binds to the P position (Eq. 13-6, step a) and that the E164 carboxyl group donates a proton to the intermediate enolate anion in step b.28 The hydroxyl group... 

The different conformational behavior of the azobenzoyl- and the azobenzenesul-fonyl-L-lysine polymers was explained on the basis that the monomeric units VI may interact with HFP differently than units V do (Scheme 4). The strongly proto-nating solvent HFP (pKa = 9.30) 36 is known to form electrostatic complexes with various organic compounds, including amines and dimethylsulfoxide 1371 on the other hand, sulfonamides are significantly protonated in acid media 38 so it may be presumed that protonation and formation of electrostatic complexes can occur for azobenzenesulfonyl-L-lysine residues, as well. In HFP therefore, polypeptides of structure V can adopt the ordered a-helix structure, while polypeptides of structure VI should be forced by the electrostatic interactions arising from complexation with HFP to adopt a disordered conformation. 

Oxidation of NADH begins with complex I, also termed NADH dehydrogenase or NADH ubiquinone oxidoreductase. It contains 25 polypeptide chains, flavine mononucleotide (FMN), and several iron-sulfur centers. The function of this complex is to reduce a substance called ubiquinone (UQ or CoQ), whose structure is shown in Figure 17.5. UQ is not protein bound and can move about freely. In the process of reducing UQ, the NADH is oxidized to NAD+. It is now accepted that in complex I, NADH first reduces FMN, and the resulting FMNH2 then transfers its electrons through at least three iron-sulfur centers to UQ. As the electrons pass from NADH to UQ, two to four protons are extruded from the mitochondrial matrix across the inner membrane. 

ATP synthase activity can be restored by adding back the F] complex to the depleted membranes. The F[ complexes bind to membrane channels known as the F complex, which are also composed of multiple subunits. The polypeptides of the F0 component are very hydrophobic and form a proton transport channel through the membrane, which links the proton gradient to ATP synthesis. This channel appears to be lined with hydrophilic residues such as seryl, threonyl and carboxyl groups. The stalk that connects the F, to the F complex comprises one copy each of the polypeptide known as the oligomycin-sensitivity-conferring protein (OSCP) and another protein known as F6. 

Step 1 Complex I. Complex I is the NADFI dehydrogenase complex consisting of more than 40 polypeptides. It accepts electrons from NADFI and passes them through flavin and iron-sulfur centers to the electron carrier ubiquinone (Q). By using one NADFI (= 2 electrons), this reaction pumps the 4 protons (2 protons/ electron) from the matrix to the intermembrane space. 

Step 2 Complex III. The cytochrome b-c 1 complex (Complex III) consists of at least 11 polypeptides and functions as a dimer. It accepts electrons from ubiquinone and transfers them to the next carrier, cytochrome c (CytC). This reaction pumps four protons (2 protons/electron) to the intermembrane space. 

Chloroplast ATP synthase is a well-defined complex which may be solubilized fi om thylakoid membranes by treatment with octylglucoside and cholate and purified by ammonium sulphate fi actionation and sucrose density gradient centrifugation [140]. The complex is composed of two assemblies of polypeptides CFj, a peripheral membrane complex, which may be washed fi-om thylakoid membranes with EDTA and which shows latent ATPase activity, and CFg, the intrinsic membrane sector, which translocates protons across the thylakoid membrane. 

This evidence has been reinforced recently by an elegant x-ray crystallographic study of the complex formed by trypsin and a polypeptide trypsin inhibitor of the bovine pancreas (25). The complex has proven to be a tetrahedral adduct which is stabilized by hydrogen bonds between the enzyme and the leaving group and by the inability of His-57 (see later) to assume a conformation which would enable it to protonate the leaving group. 

The protease exists as a homodimer. Each 99-residue monomer contains 10 j3-strands and the dimer is stabilized by a four-stranded antiparallel jS-sheet formed by the N- and C-terminal strands of each monomer. The active site of the enzyme is formed at the interface, where each monomer contributes a catalytic triad (Asp2 -Thr2 -Gly ) that is responsible for cleavage of the protease substrates. The "flap region" is located above the reactive site and is formed by a hairpin from each monomer of two antiparallel j3-strands joined by a j8-turn. There is little difference between the solution and crystal structures of protease-inhibitor complexes, except in those regions where the polypeptide chain is disordered. However, experiments in solution have allowed access to parameters that are not directly accessible from crystal data. These parameters, such as the amplitude and frequency of backbone dynamics, the protonation states of the catalytic aspartate residues, and the rate of monomer interchange, are essential in understanding the interaction of HIV protease with potent inhibitors. 

---