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Research describes the construction of a family of water-soluble heme protein maquettes consisting of four-alpha-helix bundles with heme-binding sites and study the influence of various micro- and macroenvironmental factors incorporated into the structures. Data show the manipulations lead to a diversity in the heme reduction midpoint potential measured.

Inhibitors and their effect on the ubiquinone binding capacity of the Rhodobacter capsulatus cytochrome bc(sub.1) complex primary energy conversion site are discussed. Stoichiometry of ubiquinone/ubihydroquinone occupancy is a key factor in the primary conversion (Q(sub.O)) site function in the cytochrome bc(sub.1) complex. The Q(sub.O) site double occupancy model, that in which diphenylamine (DPA) and methoxyacrylate (MOA) stilbene inhibit by displacing one and only one of the Q(sub.O) site ubiquinones, would seem to be valid.

Research was conducted to examine the nature of diphenylamine (DPA) inhibition of the cyt bc1 complex in cytoplasmic membranes of the photosynthetic prokaryote Rhodobacter capsulatus. Results indicate that DPA behaves as a noncompetitive inhibitor of the Q(sub o) site ubiquinone occupancy. Findings suggest that DPA can displace the more weakly bound of the two ubiquinones in the site.

The second-order rate constant for cytochrome c reduction evaluated using stopped-flow spectrophotometry depends solely on the rate of complex formation. Suppression of the pre-steady-state reduction of cytochrome c by zinc-substituted cytochrome c or ferrocytochrome c allows the determination of a K(sub d) for the catalytically competent complex. The suppression is slight, suggesting that electron transfer from the complex is possible and that other transfer sites are present. The effect of tight binding and many sites gives a low K(sub m) and high a K(sub cat) for flavocytochrome b2.

The role of the hinge region in intraprotein electron transfer was determined by examining the effects of deletions in the interdomain hinge region of L-lactate:cytochrome c oxidoreductase (flavocytochrome b2). Three mutants were constructed by site-directed mutagenesis using the oligonucleotides of 327N: Hdelta3, Hdelta6 and Hdelta9. Intraprotein electron transfer was monitored by steady-state and stopped-flow kinetic analyses. Kinetic data for the three mutants are presented and discussed.

Interdomain electron transfer reaction to maintain the structural hinge integrity is performed by two domains of flavocytochrome connected by the hinge peptide. The construction of H-delta3 mutant enzyme, which has three amino acid deletion near the hinge region, reveals the structure and function of the peptide hinges. Steady state kinetics is used to study inter- and intra-electron transfer in flavocytochrome.

General anesthetics have been reported to alter the functions of G protein coupled receptor (GPCR) signaling systems. To determine whether these effects might be mediated by direct binding interactions with the GPCR or its associated G protein, we studied the binding character of halothane on mammalian rhodopsin, structurally the best understood GPCR, by using direct photoaffinity labeling with [(14)C]halothane. In the bleached bovine rod disk membranes (RDM), opsin and membrane lipids were dominantly photolabeled with [(14)C]halothane, but none of the three G protein subunits were labeled. In opsin itself, halothane labeling was inhibited by unlabeled halothane with an IC(50) of 0.9 mM and a Hill coefficient of -0.8. The stoichiometry was 1.1:1.0 (halothane:opsin molar ratio). The IC(50) values of isoflurane and 1-chloro-1,2, 2-trifluorocyclobutane were 5.0 and 15 mM, respectively. Ethanol had no effect on opsin labeling by halothane. A nonimmobilizer, 1, 2-dichlorohexafluorocyclobutane, inhibited halothane labeling by 50% at 0.05 mM. The present results demonstrate that halothane binds specifically and selectively to GPCRs in the RDM. The absence of halothane binding to any of the G protein subunits strongly suggests that the functional effects of halothane on GPCR signaling systems are mediated by direct interactions with receptor proteins.

Diphenylamine (DPA), a known inhibitor of polyene and isoprene biosynthesis, is shown to inhibit flash-activatable electron transfer in photosynthetic membranes of Rhodobacter capsulatus. DPA is specific to the QO site of ubihydroquinone:cytochrome c oxidoreductase, where it inhibits not only reduction of the [2Fe-2S]2+ cluster in the FeS subunit and subsequent cytochrome c reduction but also heme bL reduction in the cytochrome b subunit. In both cases, the kinetic inhibition constant (Ki) is 25 +/- 10 microM. A novel aspect of the mode of action of DPA is that complete inhibition is established without disturbing the interaction between the reduced [2Fe-2S]+ cluster and the QO site ubiquinone complement, as observed from the electron paramagnetic resonance (EPR) spectral line shape of the reduced [2Fe-2S] cluster, which remained characteristic of two ubiquinones being present. These observations imply that DPA is behaving as a noncompetitive inhibitor of the QO site. Nevertheless, at higher concentrations (>10 mM), DPA can interfere with the QO site ubiquinone occupancy, leading to a [2Fe-2S] cluster EPR spectrum characteristic of the presence of only one ubiquinone in the QO site. Evidently, DPA can displace the more weakly bound of the two ubiquinones in the site, but this is not requisite for its inhibiting action.

Homogeneous 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from rat liver cytosol catalyzes the NAD(P)+-dependent oxidation of non-K-region trans-dihydrodiols of polycyclic aromatic hydrocarbons, many of which are proximate carcinogens. These reactions proceed with Km values in the millimolar range to yield highly reactive o-quinones that can be trapped as thioether adducts [Smithgall, T. E., Harvey, R. G., & Penning, T. M. (1988) J. Biol. Chem. 263, 1814-1820]. The enzymatically generated o-quinones, e.g., naphthalene-1,2-dione and benzo[a]pyrene-7,8-dione are potent inhibitors of the dehydrogenase, yielding IC50 values of 5.0 and 10.0 microM, respectively. Naphthalene-1,2-dione was found to be an efficient irreversible inhibitor of the enzyme and can inactivate equimolar concentrations of the dehydrogenase, yielding a t 1/2 for the enzyme of 10 s or less. By contrast (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene promotes a slower inactivation of the dehydrogenase, yielding a Kd of 70 microM and a limiting rate constant that corresponds to a t 1/2 at saturation of 23.2 min. Inactivation by this dihydrodiol has an obligatory requirement for NADP+. Examination of the kcat for the oxidation of (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene yields a partition ratio for the dihydrodiol of 200,000, suggesting that alkylation from the parent dihydrodiol is a rare occurrence. Benzo[a]pyrene-7,8-dione, which is the product of the enzymatic oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, also promotes a time- and concentration-dependent inactivation of the dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)

High-resolution proton NMR spectroscopy has been used to monitor the internal pH of chromaffin granule ghosts during Ca2+ influx through the membrane. For this purpose, ghosts were prepared by lysing and resealing chromaffin granules in a medium containing the disodium-ethylenediaminetetraacetic acid complex (Na2.EDTA). Uncomplexed EDTA and Ca.EDTA give rise to distinct sets of methylene peaks in the proton NMR spectrum. Free EDTA titrates with a pK near 6.6 in deuterated media; the chemical shifts that accompany titration have been used to monitor intravesicular pH changes which occur inside chromaffin granule ghosts as a result of ATPase activity and deprotonation of EDTA during Ca2+ influx and complex formation. ATPase activity results in an NMR-detectable proton gradient which is dissipated by nigericin. Experiments monitoring Ca2+ uptake showed that protons which are liberated inside ghosts as a result of Ca.EDTA complex formation are not extruded from the ghosts via a process coupled to Ca2+ entry. This suggests that the Ca2+ transport system of the chromaffin granule membrane occurs without concurrent proton antiport and is not directly coupled energetically to the transmembrane pH gradient.