Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone, in a reaction opposite to that catalyzed by the related enzyme succinate:quinone reductase (succinate dehydrogenase). In the previously determined structure of QFR from Wolinella succinogenes, the site of fumarate reduction in the flavoprotein subunit A of the enzyme was identified, but the site of menaquinol oxidation was not. In the crystal structure, the acidic residue Glu-66 of the membrane spanning, diheme-containing subunit C lines a cavity that could be occupied by the substrate menaquinol. Here we describe that, after replacement of Glu-C66 with Gln by site-directed mutagenesis, the resulting mutant is unable to grow on fumarate and the purified enzyme lacks quinol oxidation activity. X-ray crystal structure analysis of the Glu-C66 3 Gln variant enzyme at 3.1-Å resolution rules out any major structural changes compared with the wild-type enzyme. The oxidationreduction potentials of the heme groups are not significantly affected. We conclude that Glu-C66 is an essential constituent of the menaquinol oxidation site. Because Glu-C66 is oriented toward a cavity leading to the periplasm, the release of two protons on menaquinol oxidation is expected to occur to the periplasm, whereas the uptake of two protons on fumarate reduction occurs from the cytoplasm. Thus our results indicate that the reaction catalyzed by W. succinogenes QFR generates a transmembrane electrochemical potential.

Flavin adenine dinucleotide (FAD) and three different flavoproteins in aqueous solution were subjected to redox-triggered Fourier transform infrared difference spectroscopy. The acquired vibrational spectra show a great number of positive and negative peaks, pertaining to the oxidized and reduced state of the molecule, respectively. Density functional theory calculations on the B3LYP/6-31G(d) level were employed to assign several of the observed bands to vibrational modes of the isoalloxazine moiety of the flavin cofactor in both its oxidized and, for the first time, its reduced state. Prominent modes measured for oxidized FAD include nu(C(4)=O) and nu(C(2)=O) at 1716 and 1674 cm(-1), respectively, nu(C(4a)=N(5)) at 1580 cm(-1), and nu(C(10a)=N(1)) at 1548 cm(-1). Measured modes of the reduced form of FAD include nu(C(2)=O) at 1692 cm(-1), nu(C(4)=O) at 1634 cm(-1), and nu(C(4a)=C(10a)) at 1600 cm(-1). While the overall shape of the enzyme spectra is similar to the shape of the spectrum of free FAD, there are numerous differences in detail. In particular, the nu(C=N) modes of the flavin exhibit frequency shifts in the protein-bound form, most prominently for pyruvate oxidase where nu(C(10a)=N(1)) downshifts by 14 cm(-1) to 1534 cm(-1). The significance of this shift and a possible explanation in connection with the bent conformation of the flavin cofactor in this enzyme are discussed.

The cytochrome bc(1) complex from Paracoccus denitrificans and soluble fragments of its cytochrome c(1) and Rieske ISP subunits are characterized by a combined approach of protein electrochemistry and FTIR difference spectroscopy. The FTIR difference spectra provide information about alterations in the protein upon redox reactions: signals from the polypeptide backbone, from the cofactors, and from amino acid side chains. We describe typical modes for conformational changes in the polypeptide and contributions of different secondary structure elements. Signals attributed to the different cofactors can be presented on the basis of selected potential steps. Modes associated with bound quinone are identified by comparison with spectra of quinone in solution at 1656, 1642, and 1610 cm(-1) and between 1494 and 1388 cm(-1), as well as at 1288 and 1262 cm(-1). Signals originating from the quinone bound at the Q(o) site can be distinguished. On the basis of the infrared data, the total quinone concentration is determined to be 2.6-3.3 quinones per monomer, depending on preparation conditions. The balance of evidence supports the double-occupancy model. Interestingly, the amplitude of the band at 1746 cm(-1) increases with quinone content, reflecting a protonation reaction of acidic groups. In this context, the involvement of glutamates and/or aspartates in the vicinity of the Q(o) site is discussed on the basis of recently determined crystal structures.

In this study a combined electrochemical and FTIR spectroscopic approach was applied to monitor the binding of stigmatellin, a Q(o) site inhibitor of the cytochrome bc(1) complex from Saccharomyces cerevisiae. Natural stigmatellin A induced clear shifts in the redox-induced FTIR difference spectra. For data interpretation a stigmatellin derivative (UST) with the conjugated trienes replaced by an aliphatic tail was synthesized, and the carbonyl group shown in crystal structures to interact with His181, the [2Fe-2S] ligand of the Rieske, was specifically (13)C labeled. Electrochemically induced FTIR difference spectra of the inhibitors in CH(3)OD were obtained and revealed signals characteristic for the oxidized and reduced forms of the labeled and unlabeled compounds. On the basis of signals from the inhibitors alone, the binding of the inhibitor to the bc(1) complex was monitored. Direct evidence for the interaction of the carbonyl group with the protein was provided by the observed shift of the nu(C=O) vibrational mode of about 10 cm(-1). In addition, redox-dependent reorganizations of the protein were identified, including protonation changes of acidic residues at 1746 and 1734 cm(-1). The conformational changes observed upon inhibitor binding are discussed with respect to the crystal structures and proposed mechanistic models [Hunte, C., Koepke, J., Lange, C., Rossmanith, T., and Michel, H. (2000) Structure 8, 669-684; Palsdottir, H., Lojero, C. G., Trumpower, B. L., and Hunte, C. (2003) J. Biol. Chem. 278, 31303-31311].

The protonation state of residues around the Qo binding site of the cytochrome bc1 complex from Paracoccus denitrificans and their interaction with bound quinone(s) was studied by a combined electrochemical and FTIR difference spectroscopic approach. Site‐directed mutations of two groups of conserved residues were investigated: (a) acidic side chains located close to the surface and thought to participate in a water chain leading up to the heme bL edge, and (b) residues located in the vicinity of this site. Interestingly, most of the mutants retain a high degree of catalytic activity. E295Q, E81Q and Y297F showed reduced stigmatellin affinity. On the basis of electrochemically induced FTIR difference spectra, we suggest that E295 and D278 are protonated in the oxidized form or that their mutation perturbs protonated residues. Mutations Y302, Y297, E81 and E295, directly perturb signals from the oxidized quinone and of the protein backbone. By monitoring the interaction with the inhibitor stigmatellin for the wild‐type enzyme at various redox states, interactions of the bound stigmatellin with amino acid side chains such as protonated acidic residues and the backbone were observed, as well as difference signals arising from the redox active inhibitor itself and the replaced quinone. The infrared difference spectra of the above Qo site mutations in the presence of stigmatellin confirm the previously established role of E295 as a direct interaction partner in the enzyme from P. denitrificans as well. The protonated residue E295 is proposed to change the hydrogen‐bonding environment upon stigmatellin binding in the oxidized form, and is deprotonated in the reduced form. Of the residues located close to the surface, D278 remains protonated and unperturbed in the oxidized form but its frequency shifts in the reduced form. The mechanistic implications of our observations are discussed, together with previous inhibitor binding data, and referred to the published X‐ray structures.

Quinones and quinoproteins are essential redox components and enzymes in biological systems. Here, we report the de novo design, synthesis, and properties of model four-alpha-helix bundle quinoproteins. The proteins were designed and constructed from three different helices with 21 or 22 amino acid residues by chemoselective ligation to a cyclic decapeptide template. A free cysteine unit is placed at the hydrophobic core of the protein for binding of ubiquinone-0 and menaquinone-0 through a thioether bond. The quinoproteins with molecular weights of 11-12 kDa were characterized by electrospray ionization mass spectrometry, UV/Vis spectroscopy, size-exclusion chromatography, circular dichroism measurements, (1)H NMR spectroscopy, cyclic voltammetry, and redox-induced FTIR difference spectroscopy. The midpoint redox potentials at pH 8 in aqueous solution E(m,8) of thioether conjugates with N-acetyl cysteine methyl ester were 89 mV and -63 mV and with a synthetic protein 229 mV and 249 mV versus standard hydrogen electrode (SHE) for ubiquinone-0 and menaquinone-0, respectively. Detailed redox-induced FTIR difference spectroscopic studies of the model compounds and quinoproteins show the special resonance features for C=O bands at 1656-1660 and 1655-1665 cm(-1) due to the sulfur substitution to ubiquinone-0 and menaquinone-0, respectively. The construction of model quinoproteins represents a significant step toward more complex artificial redox systems.

In pyruvate oxidase (POX) from Lactobacillus plantarum, valine 265 participates in binding the cofactor FAD and is responsible for the strained conformation of its isoalloxazine moiety that is visible in the crystal structure of POX. The contrasting effects of the conservative amino acid exchange V265A on the enzyme's catalytic properties, cofactor affinity, and protein structure were investigated. The most prominent effect of the exchange was observed in the 2.2 A crystal structure of the mutant POX. While the overall structures of the wild-type and the variant are similar, flavin binding in particular is clearly different. Local disorder at the isoalloxazine binding site prevents modeling of the complete FAD cofactor and two protein loops of the binding site. Only the ADP moiety shows well-defined electron density, indicating an "anchor" function for this part of the molecule. This notion is corroborated by competition experiments where ADP was used to displace FAD from the variant enzyme. Despite the fact that the affinity of FAD binding in the variant is reduced, the catalytic properties are very similar to the wild-type, and the redox potential of the bound flavin is the same for both proteins. The rate of electron transfer toward the flavin during turnover is reduced to one-third compared to the wild-type, but k(cat) remains unchanged. Redox-triggered FTIR difference spectroscopy of free FAD shows the nu(C(10a)=N(1)) band at 1548 cm(-)(1). In POX-V265A, this band is found at 1538 cm(-)(1) and thus shifted less strongly than in wild-type POX where it is found at 1534 cm(-)(1). Taking these observations together, the conservative exchange V265A in POX has a surprisingly small effect on the catalytic properties of the enzyme, whereas the effect on the three-dimensional structure is rather big.

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