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P L Dutton

Publications and source records attributed to P L Dutton.

At least 37 records · Page 2Linked to original sources
PubMed1999

Histidine placement in de novo-designed heme proteins.

The effects of histidine residue placement in a de novo-designed four-alpha-helix bundle are investigated by placement of histidine residues at coiled coil heptad a positions in two distinct heptads and at each position within a single heptad repeat of our prototype heme protein maquette, [H10H24]2 [[Ac-CGGGELWKL x HEELLKK x FEELLKL x HEERLKK x L-CONH2]2]2 composed of a generic (alpha-SS-alpha)2 peptide architecture. The heme to peptide stoichiometry of variants of [H10H24]2 with either or both histidines on each helix replaced with noncoordinating alanine residues ([H10A24]2, [A10H24]2, and [A10A24]2) demonstrates the obligate requirement of histidine for biologically significant heme affinity. Variants of [A10A24]2, [[Ac-CGGGELWKL x AEELLKK x FEELLKL x AEERLKK x L-CONH2]2]2, containing a single histidine per helix in positions 9 to 15 were evaluated to verify the design based on molecular modeling. The bis-histidine site formed between heptad positions a at 10 and 10' bound ferric hemes with the highest affinity, Kd1 and Kd2 values of 1.5 and 800 nM, respectively. Placement of histidine at position 11 (heptad position b) resulted in a protein that bound a single heme with moderate affinity, Kd1 of 9.5 microM, whereas the other peptides had no measurable apparent affinity for ferric heme with Kd1 values >200 microM. The bis-histidine ligation of heme to [H10A24]2 and [H11A24]2 was confirmed by electron paramagnetic resonance spectroscopy. The protein design rules derived from this study, together with the narrow tolerances revealed, are applicable for improving future heme protein designs, for analyzing the results of randomized heme protein combinatorial libraries, as well as for implementation in automated protein design.

Amino Acid Sequence↗
PubMed1998

Functionalized de novo designed proteins: mechanism of proton coupling to oxidation/reduction in heme protein maquettes.

Proton exchange with aqueous media coupled to heme oxidation/reduction is commonly seen but not understood in natural cytochromes. Our synthetic tetrahelix bundle heme protein maquettes successfully reproduce natural proton coupling to heme oxidation/reduction. Potentiometry reveals major pK shifts from 4.2 to 7.0 and from 9.4 to 10.3 in the maquette-associated acid/base group(s) upon heme reduction. Consequently, a 210 mV decrease in the heme redox potential is observed between the two extremes of pH. Potentiometry with resonance Raman and FTIR spectroscopy performed over a wide pH range strongly implicates glutamate side chains as the source of proton coupling below pH 8.0, whereas lysine side chains are suggested above pH 8.0. Remarkably, the pK values of several glutamates in the maquette are elevated from their solution value (4.4) to values as high as 7.0. It is suggested that these glutamates are recruited into the interior of the bundle as part of a structural rearrangement that occurs upon heme binding. Glutamate to glutamine variants of the prototype protein demonstrate that removal of the glutamate closest to the heme diminishes but does not abolish proton exchange. It is necessary to remove additional glutamates before pH-independent heme oxidation/reduction profiles are achieved. The mechanism of redox-linked proton coupling appears to be rooted in distributed partial charge compensation, the magnitude of which is governed by the dielectric distance between the ferric heme and acid/base side chains. A similar mechanism is likely to exist in native redox proteins which undergo charge change upon cofactor oxidation/reduction.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of a two-subunit cytochrome b-c1 subcomplex from Rhodobacter capsulatus and reconstitution of its ubihydroquinone oxidation (Qo) site with purified Fe-S protein subunit.

The presence of a two-subunit cytochrome (cyt) b-c1 subcomplex in chromatophore membranes of Rhodobacter capsulatus mutants lacking the Rieske iron-sulfur (Fe-S) protein has been described previously [Davidson, E., Ohnishi, T., Tokito, M., and Daldal, F. (1992) Biochemistry 31, 3351-3358]. Here, this subcomplex was purified to homogeneity in large quantities, and its properties were characterized. As expected, it contained stoichiometric amounts of cyt b and cyt c1 subunits forming a stable entity devoid of the Fe-S protein subunit. The spectral and thermodynamic properties of its heme groups were largely similar to those of a wild-type bc1 complex, except that those of its cyt bL heme were modified as revealed by EPR spectroscopy. Dark potentiometric titrations indicated that the redox midpoint potential (Em7) values of cytochromes bH, bL, and c1 were very similar to those of a wild-type bc1 complex. The purified b-c1 subcomplex had a nonfunctional ubihydroquinone (UQH2) oxidation (Qo) site, but it contained an intact ubiquinone (UQ) reductase (Qi) site as judged by its ability to bind the Qi inhibitor antimycin A, and by the presence of antimycin A sensitive Qi semiquinone. Interestingly, its Qo site could be readily reconstituted by addition of purified Fe-S protein subunit. Reactivated complex exhibited myxothiazol, stigmatellin, and antimycin A sensitive cyt c reductase activity and an EPR gx signal comparable to that observed with a bc1 complex when the Qo site is partially occupied with UQ/UQH2. However, a mutant derivative of the Fe-S protein subunit lacking its first 43 amino acid residues was unable to reactivate the purified b-c1 subcomplex although it could bind to its Qo site in the presence of stigmatellin. These findings demonstrated for the first time that the amino-terminal membrane-anchoring domain of the Fe-S protein subunit is necessary for UQH2 oxidation even though its carboxyl-terminal domain is sufficient to provide wild-type-like interactions with stigmatellin at the Qo site of the bc1 complex.

Binding Sites↗
PubMed1998

Design, synthesis, and characterization of a photoactivatable flavocytochrome molecular maquette.

We report the construction of a synthetic flavo-heme protein that incorporates two major physiological activities of flavoproteins: light activation of flavin analogous to DNA photolyase and rapid intramolecular electron transfer between the flavin and heme cofactors as in several oxidoreductases. The functional tetra-alpha-helix protein comprises two 62-aa helix-loop-helix subunits. Each subunit contains a single cysteine to which flavin (7-acetyl-10-methylisoalloxazine) is covalently attached and two histidines appropriately positioned for bis-his coordination of heme cofactors. Both flavins and hemes are situated within the hydrophobic core of the protein. Intramolecular electron transfer from flavosemiquinone generated by photoreduction from a sacrificial electron donor in solution was examined between protoporphyrin IX and 1-methyl-2-oxomesoheme XIII. Laser pulse-activated electron transfer from flavin to meso heme occurs on a 100-ns time scale, with a favorable free energy of approximately -100 meV. Electron transfer from flavin to the lower potential protoporphyrin IX, with an unfavorable free energy, can be induced after a lag phase under continuous light illumination. Thus, the supporting peptide matrix provides an excellent framework for the positioning of closely juxtaposed redox groups capable of facilitating intramolecular electron transfer and begins to clarify in a simplified and malleable system the natural engineering of flavoproteins.

Amino Acid Sequence↗
PubMed1998

Non-inhibiting perturbation of the primary energy conversion site (Qo site) in Rhodobacter capsulatus ubihydroquinone: cytochrome c oxidoreductase (cytochrome bc1 complex).

Ethanol added to Rhodobacter capsulatus chromatophore membranes containing the cytochrome bc1 complex effectively uncouples the sensitivity of the [2Fe-2S] cluster EPR spectrum to the number and redox state of ubiquinone/ubihydroquinone within the Qo site. Ethanol has no effect upon the rate of catalysis, leading to a non-inhibiting perturbation of cytochrome bc1 function. We suggest that displacement occurs by ethanol acting from the aqueous phase to successfully compete with the Qo site ubiquinones and water to hydrogen bond the N(epsilon)H atom(s) of the coordinating [2Fe-2S] cluster histidines.

Binding Sites↗
PubMed1998

A reductant-induced oxidation mechanism for complex I.

A model for energy conversion in Complex I is proposed that is a conservative expansion of Mitchell's Q-cycle using a simple mechanistic variation of that already established experimentally for Complex III. The model accommodates the following proposals. (1) The large number of flavin and iron-sulfur redox cofactors integral to Complex I form a simple but long electron transfer chain guiding submillisecond electron transfer from substrate NADH in the matrix to the [4Fe-4S] cluster N2 close to the matrix-membrane interface. (2) The reduced N2 cluster injects a single electron into a ubiquinone (Q) drawn from the membrane pool into a nearby Qnz site, generating an unstable transition state semiquinone (SQ). The generation of a SQ species is the primary step in the energy conversion process in Complex I, as in Complex III. In Complex III, the SQ at the Qo site near the cytosolic side acts as a strong reductant to drive electronic charge across the membrane profile via two hemes B to a Qi site near the matrix side. We propose that in Complex I, the SQ at the Qnz site near the matrix side acts as a strong oxidant to pull electronic charge across the membrane profile via a quinone (Qny site) from a Qnx site near the cytosolic side. The opposing locations of matrix side Qnz and cytosolic side Qo, together with the opposite action of Qnz as an oxidant rather than a reductant, renders the Complex I and III processes vectorially and energetically complementary. The redox properties of the Qnz and Qo site occupants can be identical. (3) The intervening Qny site of Complex I acts as a proton pumping element (akin to the proton pump of Complex IV), rather than the simple electron guiding hemes B of Complex III. Thus the transmembrane action of Complex I doubles to four (or more) the number of protons and charges translocated per NADH oxidized and Q reduced. The Qny site does not exchange with the pool and may even be covalently bound. (4) The Qnx site on the cytosol side of Complex I is complementary to the Qi site on the matrix side of Complex III and can have the same redox properties. The Qnx site draws QH2 from the membrane pool to be oxidized in two single electron steps. Besides explaining earlier observations and making testable predictions, this Complex I model re-establishes a uniformity in the mechanisms of respiratory energy conversion by using engineering principles common to Complexes III and IV: (1) all the primary energy coupling reactions in the different complexes use oxygen chemistry in the guise of dioxygen or ubiquinone, (2) these reactions are highly localized structurally, utilizing closely placed catalytic redox cofactors, (3) these reactions are also highly localized energetically, since virtually all the free energy defined by substrates is conserved in the form of transition state that initiates the transmembrane action and (4) all complexes possess apparently supernumerary oxidation-reduction cofactors which form classical electron transfer chains that operate with high directional specificity to guide electron at near zero free energies to and from the sites of localized coupling.

Electron Transport↗
PubMed1998

Effect of four helix bundle topology on heme binding and redox properties.

We have designed two alternative four helix bundle protein scaffold topologies for maquette construction to examine the effect of helix orientation on the heme binding and redox properties of our prototype heme protein maquette, (alpha-SS-alpha)2, previously described as H10H24 [Robertson, D. E., Farid, R. S., Moser, C. C., Mulholland, S. E., Pidikiti, R., Lear, J. D., Wand, A. J., DeGrado, W. F., and Dutton, P. L. (1994) Nature 368, 425]. Conversion of the disulfide-bridged di-alpha-helical monomer of (alpha-SS-alpha)2 into a single polypeptide chain results in topological reorientation of the helix dipoles and side chains within a 62 amino acid helix-loop-helix monomer, (alpha-l-alpha), which self-associates to form (alpha-l-alpha)2. Addition of an N-terminal cysteine residue to (alpha-l-alpha) with subsequent oxidation yields a 126 amino acid single molecule four helix bundle, (alpha-l-alpha-SS-alpha-l-alpha). Gel permeation chromatography demonstrated that (alpha-SS-alpha)2 and (alpha'-SS-alpha')2, a uniquely structured variant of the prototype, as well as (alpha-l-alpha)2 and (alpha'-l-alpha')2 assemble into distinct four helix bundles as designed, whereas (alpha-l-alpha-SS-alpha-l-alpha) elutes as a monomeric four alpha-helix bundle. Circular dichroism (CD) spectroscopy proves that these peptides are highly alpha-helical, and incorporation of four hemes has little effect on the helical content of the secondary structure. Four heme dissociation constants were evaluated by UV-visible spectroscopy and ranged from the 15 nM to 25 microM range for each of the peptides. The presence of Cotton effects in the visible CD illustrated that the hemes reside within the protein architecture. The equilibrium redox midpoint potentials (Em8) of the four bound hemes in each peptide are between -100 and -280 mV, as determined by redox potentiometry. The heme affinity and spectroelectrochemical properties of the hemes bound to (alpha-l-alpha)2 and (alpha-l-alpha-SS-alpha-l-alpha) are similar to those of the prototype, (alpha-SS-alpha)2, and to bis-histidine ligated b-type cytochromes, regardless of the global architectural changes imposed by these topological rearrangements. The hydrophobic cores of these peptides support local electrostatic fields which result in nativelike heme chromophore properties (spectroscopy, elevated reduction potentials, heme-heme charge interaction, and reactivity with exogenous diatomics) illustrating the utility of these non-native peptides in the study of metalloproteins.

Amino Acid Sequence↗
PubMed1998

A designed cavity in the hydrophobic core of a four-alpha-helix bundle improves volatile anesthetic binding affinity.

The structural features of protein binding sites for volatile anesthetics are being explored using a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. Earlier work has demonstrated that a prototype hydrophobic core is capable of binding the volatile anesthetic halothane. Exploratory work on the design of an improved affinity anesthetic binding site is presented, based upon the introduction of a simple cavity into a prototype (alpha 2)2 four-alpha-helix bundle by replacing six core leucines with smaller alanines. The presence of such a cavity increases the affinity (Kd = 0.71 +/- 0.04 mM) of volatile anesthetic binding to the designed bundle core by a factor of 4.4 as compared to an analogous bundle core lacking such a cavity (Kd = 3.1 +/- 0.4 mM). This suggests that such packing defects present on natural proteins are likely to be occupied by volatile general anesthetics in vivo. Replacing six hydrophobic core leucine residues with alanines results in a destabilization of the folded bundle by 1.7-2.7 kcal/mol alanine, although the alanine-substituted bundle still exhibits a high degree of thermodynamic stability with an overall folded conformational delta GH2O = 14.3 +/- 0.8 kcal/mol. Covalent attachment of the spin label MTSSL to cysteine residues in the alanine-substituted four-alpha-helix bundle indicates that the di-alpha-helical peptides dimerize in an anti orientation. The rotational correlation time of the four-alpha-helix bundle is 8.1 +/- 0.5 ns, in line with earlier work on similar peptides. Fluorescence, far-UV circular dichroism, and Fourier transform infrared spectroscopies verified the hydrophobic core location of the tryptophan and cysteine residues, showing good agreement between experiment and design. These small synthetic proteins may prove useful for the study of the structural features of small molecule binding sites.

Amino Acid Sequence↗
PubMed1997

Substitutions at position 146 of cytochrome b affect drastically the properties of heme bL and the Qo site of Rhodobacter capsulatus cytochrome bc1 complex.

The cytochrome (cyt) b subunit of ubihydroquinone: cytochrome c oxidoreductase (bc1 complex) contains four invariant glycine (G) residues proposed to be essential for proper packing of the high and low potential (bH and bL) hemes of the bc1 complex. One of these residues, G146 located in the transmembrane helix C of cyt b of Rhodobacter capsulatus, was substituted with A and V using site-directed mutagenesis, and the effects of these substitutions on the properties of the ubiquinone oxidation (Qo) site and heme bL of the bc1 complex were analyzed. The mutants G146A and V produced properly assembled but catalytically defective bc1 complexes that are unable to support photosynthetic growth. The steady-state ubihydroquinone: cytochrome c reductase activities of the mutant complexes were about one-tenth of that of a parental strain overproducing the wild-type enzyme. Similarly, their light-activated single turnover rates were significantly lower than those of a wild-type complex. The dark potentiometric titrations revealed no significant changes in the redox midpoint potentials (Em.7) of the high (bH) and low (bL) potential hemes of cyt b in both G146A and V mutants. However, EPR spectroscopy of the [2Fe-2S] cluster of the bc1 complex indicated that the Qo site of the mutant enzymes were unoccupied. Moreover, the gz signal of heme bL, but not that of heme bH, was modified both in G146A and V, suggesting that the geometry of its ligands has been distorted. These findings indicate that this region of cyt b must be well packed around heme bL since even a slight increase in the size of the amino acid side chain at position 146 (such as G to A) greatly perturbs the spatial conformation of heme bL, alters substrate accessibility and binding to the Qo site, and renders the bc1 complex inactive.

Alanine↗
PubMed1997

Global topology & stability and local structure & dynamics in a synthetic spin-labeled four-helix bundle protein.

A maleimide nitroxide spin-label (MAL-6) linked to a cysteine in the hydrophobic core and a coproporphyrin I (CP) appended on the N-terminus of a synthetic helix-loop-helix peptide ([alpha2]) have been used to examine the designed self-association of a four-helix bundle ([alpha2]2), focusing on the bundle topology and stability and the rotational dynamics of the spin-label. Gel-permeation chromatography demonstrated that the [alpha2] peptide and the peptide modified with a spin-label ([MAL-6-alpha2]), a coproporphyrin ([CP-alpha2]) and a coproporphyrin plus a spin-label ([CP-MAL-6-alpha2]) self-associate into four helix bundles in solution as designed. Circular dichroism (CD) spectra prove that all these peptides are highly alpha-helical, confirmed for [alpha2]2 by Fourier transform infrared (FTIR) spectroscopic analysis. Electron spin resonance (ESR) spectra of the two attached maleimide spin-labels in [MAL-6-alpha2]2 shows their effective rotational correlation time (tau(c)) is 7.3 +/- 0.5 ns, consistent with that expected for the tumbling of the four helix bundle itself, indicating the labels are immobilized. The ESR spectra were also unaltered by aqueous-phase paramagnetic ions, Ni(II), demonstrating all of the spin-labels are buried within the hydrophobic core. The lack of spin-spin interaction between the buried, immobilized spin-labels indicates they are remote (> 15 A) from each other, indicating an antiparallel topology of the monomers in [MAL-6-alpha2]2. The parent [alpha2]2 and the modified [MAL-6-alpha2]2 and [CP-alpha2]2 peptides are highly stable (deltaG(H2O) approximately 25 kcal/mol) as investigated by guanidine hydrochloride denaturation curves monitored by ESR and CD spectroscopies. Guanidine hydrochloride denaturation leads to a shorter correlation time of the spin-label, tau(c) < 1 ns, approaching that of an unrestricted spin-label in solution. In contrast, trifluoroethanol caused dissociation of [MAL-6-alpha2]2 to yield two [MAL-6-alpha2] monomers with retention of secondary structure and changed the tau(c) to 2.5 +/- 0.5 ns, indicating that a significant degree of motional restriction is imposed on the spin-label by the secondary structure. The coproporphyrin probes covalently attached to the N-termini of [CP-alpha2]2 and [CP-MAL-6-alpha2]2 provided evidence that the helical monomers of both were in a parallel orientation, in contrast to the antiparallel orientation determined for [MAL-6-alpha2]2. Consequently, the ESR spectra of [MAL-6-alpha2]2 and [CP-MAL-6-alpha2]2 reveal major structural differences in the local vicinity of the spin-labels due to the topological difference between these two bundles. The ESR spectra of [CP-MAL-6-alpha2]2 contains two distinct nitroxide populations, indicating that one spin-label remains buried in the hydrophobic core and the other is excluded to solvent in this parallel topology. Alleviation of the steric interactions causing one spin-label in [CP-MAL-6-alpha2]2 to be solvent-exposed by addition of [CP-alpha2]2 results in formation of the heterodimeric [CP-alpha2]/[CP-MAL-6-alpha2], as evidenced by insertion of all the spin-labels into hydrophobic cores. The changes in global topology and local structure as evidenced by this pair of spectral probes have relatively minor effects on the course of guanidine denaturation of these bundles.

Amino Acid Sequence↗
PubMed1997

Synthesis of novel proteins.

De novo and rational protein design are progressing towards the chemical synthesis of proteins with pre-selected structure and function. The data illustrate diverse experimental and computational approaches which test our comprehension of protein structure, hydrophobic core packing and global stability, especially of coiled-coil proteins. The incorporation of biological cofactors, including hemes, as well as active sites, such as that of iron superoxide dismutase, into designed proteins provides an exciting next step towards the synthesis of proteins with enzymatic function.

Amino Acid Sequence↗
PubMed1996

Ferredoxin and ferredoxin-heme maquettes.

A 16-amino acid residue peptide derived from a consensus motif of natural ferredoxins incorporates a tetranuclear iron sulfur cluster under physiological conditions. Successful assembly of the [4Fe-4S]2+/1+ cluster within a monomeric peptide was demonstrated using size exclusion chromatography, UV-visible, visible CD, and cryogenic EPR spectroscopies. The robustness of [4Fe-4S]2+/1+ formation was tested using peptides with either the ligating cysteine exchanged for alanine or with the intervening amino acids replaced by glycine. The small size of the peptide allows for modular incorporation into more complex protein structures. In one larger structure, we describe a tetra-alpha-helix bundle that self-assembles both iron-sulfur clusters and hemes, thereby demonstrating feasibility for the general synthesis of maquettes containing multiple, juxtaposed redox cofactors. This is a motif common to the catalytic sites of native oxidoreductases.

Amino Acid Sequence↗
PubMed1996

Structural and electronic properties of the heme cofactors in a multi-heme synthetic cytochrome.

Resonance Raman, absorption, and electron paramagnetic resonance spectra are reported for a water soluble, synthetic cytochrome. The protein is a variant of the cytochrome beta maquette described by Robertson et al. [Robertson, D. E., et al. (1995) Nature 368, 425-432] and is composed of 62 amino acid residues arranged in a di-alpha-helical unit which dimerizes in solution to form a four-helix bundle. Each di-alpha-helical unit contains histidine residues at the 10,10' positions which serve as ligands to the hemes. When protoheme IX is incorporated, both hemes in the dimer are bis-ligated and low spin. The two hemes are inequivalent with respect to both binding affinity and redox properties. To investigate the properties of the heme cofactors, spectroscopic studies were conducted on peptides reconstituted with protoheme IX (PHa) and several related variants. These hemes include 2-vinyldeuteroheme (2-VDH), 4-vinyldeuteroheme (4-VDH), protoheme III (PHs), and 1-methyl-2-oxomesoheme XIII (2-OMH). Collectively, the spectroscopic studies reveal the following: (1) 2-VDH, 4-VDH, and 2-OMH bind to the protein and form bis-ligated low-spin complexes similar to PHa. The structures of the two hemes in the dimers are identical as are the immediate protein environments around the bound cofactors. These results indicate that the redox inequivalence of the two hemes is due to heme-heme electronic interactions rather than structural and/or environmental differences between the two cofactors. (2) The two hemes in the dimer are arranged in a edge-to-edge arrangement wherein the oxo group (2-OMH) or the vinyl group(s) are in the hydrophobic interface between the two units which comprise the dimer. The propionic acid tails point outward toward the hydrophilic region and extend into the solvent. (3) The PHs protein differs from the other synthetic proteins in that it contains one pentacoordinate, high-spin and one hexacoordinate, low-spin heme rather than two hexacoordinate low-spin cofactors. The open coordination site on the high-spin heme is inaccessible to exogenous imidazole but readily bind cyanide, suggesting that the alpha-helix containing the unbound histidine is nearby and partially shields the coordination site. The high-spin heme converts to low-spin at low-temperature, presumably via binding of the histidine residue on this nearby alpha-helix. It is suggested that the different behavior observed for the PHs protein is due to the fact that this heme is symmetric with respect to rotation about the alpha,gamma-axis of the macrocycle which bisects the meso-carbons between the vinyl groups and propionic acid residues. This symmetry precludes rotational isomerism about the alpha,gamma-axis to establish an unhindered fit. In contrast, all the other hemes examined contain at least one substituent smaller than a vinyl group which together with the fact that two different alpha,gamma-rotational isomers are possible for each heme in the dimer could allow these hemes to avoid the like-substituent--like-substituent heme--heme interactions of PHs. The propensity to avoid such interactions could explain the inequivalent binding properties of the two hemes in the dimer. For the PHs protein wherein these these interactions cannot be mitigated by rotation of the heme, other rearrangements of the protein must occur. These rearrangements could force the second-bound heme to assume a high-spin configuration.

Cytochromes↗
PubMed1996

Binding of the volatile anesthetic halothane to the hydrophobic core of a tetra-alpha-helix-bundle protein.

Although volatile general anesthetics interact with several proteins, little is known about the location or characteristics of the binding sites at the molecular level. A detailed structural description of how anesthetics associate with macromolecules is necessary for understanding anesthetic mechanisms of action. The recent introduction of designed synthetic proteins provides new opportunities for obtaining structural and functional information on anesthetic-protein interactions. A synthetic tetra-alpha-helix-bundle protein was used to examine the interaction of halothane with a designed protein interior. The tetra-alpha-helix-bundle comprises 124 residues in the form of two identical 62-residue di-alpha-helical peptides, held together in an all-parallel bundle by hydrophobic forces. Steady-state and time-resolved tryptophan fluorescence and circular dichroism spectroscopy were used to study the anesthetic-protein interaction. Halothane quenches bundle tryptophan fluorescence with a dissociation constant of 2.3 +/- 0.4 mM and a Hill number of 0.9 +/- 0.1. Tryptophan fluorescence decay analysis indicates that halothane quenches the protein fluorescence by a static mechanism. Circular dichroism spectroscopy revealed no change in protein secondary structure on exposure to halothane. Dissociation of the tetra-alpha-helix-bundle into 62-residue di-alpha-helical peptides by trifluoroethanol eliminated the halothane-protein interaction. The results suggest that halothane binds to the hydrophobic interior of the tetra-alpha-helix-bundle, close to the tryptophan residues. The protein tertiary and quaternary structures are required for anesthetic binding. This study demonstrates the feasibility of using synthetic tetra-alpha-helix-bundles as model anesthetic-binding proteins. The use of de novo designed bundle proteins should allow structural, energetic and functional descriptions of anesthetic-protein interactions.

Amino Acid Sequence↗
PubMed1995

Ubiquinone pair in the Qo site central to the primary energy conversion reactions of cytochrome bc1 complex.

The mechanistic heart of the ubihydroquinone-cytochrome c oxidoreductase (cyt bc1 complex) is the catalytic oxidation of ubihydroquinone (QH2) at the Qo site. QH2 oxidation is initiated by ferri-cyt c, mediated by the cyt c1 and [2Fe-2S] cluster of the cytochrome bc1 complex. QH2 oxidation in turn drives transmembrane electronic charge separation through two b-type hemes to another ubiquinone (Q) at the Qi site. In earlier studies, residues F144 and G158 of the b-heme containing polypeptide of the Rhodobacter capsulatus cyt bc1 complex were shown to be influential in Qo site function. In the present study, F144 and G158 have each been singly substituted by neutral residues and the dissociation constants measured for both Q and QH2 at each of the strong and weak binding Qo site domains (Qos and Qow). Various substitutions at F144 or G158 were found to weaken the affinities for Q and QH2 at both the Qos and Qow domains variably from zero to beyond 10(3)-fold. This produced a family of Qo sites with Qos and Qow domain occupancies ranging from nearly full to nearly empty at the prevailing approximately 3 x 10(-2) M concentration of the membrane ubiquinone pool (Qpool). In each mutant, the affinity of the Qos domain remained typically 10-20-fold higher than that of the Qow domain, as is found for wild type, thereby indicating that the single mutations caused comparable extents of the weakening at each domain. Moreover, the substitutions were found to cause similar decreases of the affinities of both Q and QH2 in each domain, thereby maintaining the Q/QH2 redox midpoint potentials (Em7) of the Qo site at values similar to that of the wild type. Measurement of the yield and rate of QH2 oxidation generated by single turnover flashes in the family of mutants suggests that the Qos and Qow domains serve different roles for the catalytic process. The yield of the QH2 oxidation correlates linearly with Qos domain occupancy (QH2 or Q), suggesting that the Qos domain exchanges Q or QH2 with the Qpool at a rate which is much slower than the time scale of turnover.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

Ion pair formation between basic residues at 144 of the Cyt b polypeptide and the ubiquinones at the Qo site of the Cyt bc1 complex.

Loci of spontaneous Qo site inhibitor resistant mutants in the cyt bc1 complex of the photosynthetic bacterium Rhodobacter capsulatus are M140, F144, G152, G158, and T163 of the cyt b polypeptide. In this report, we have studied the effects of arginine (R) substitution at these positions with a view to test for specific interactions with the [2Fe-2S] cluster, cyt bL with Qo site ubiquinone (Q), or hydroquinone (QH2). All the arginine mutants displayed severely or completely impeded photosynthetic growth resulting from dysfunctional cyt bc1 complexes. The source of dysfunction in G158R and T163R was identified by a > 1000-fold decrease in the Qo site affinity for QH2 and Q, sufficient to empty the site in the presence of the 30 mM ubiquinone pool of the chromatophore membrane; they appear similar to the class of mutants described in the preceding paper [Ding, H., Moser, C. C., Robertson, D. E., Tokito, M., Daldal, F., & Dutton, P. L (1995) Biochemistry 34, 15979-15996]. The source(s) of dysfunction of M140R and G152R is not so apparent since they possess Qo sites with normal QH2/Q affinity; they appear to be members to the class of mutants identified and characterized in the following paper [Saribaş, S., Ding, H., Dutton, P. L., & Daldal, F. (1995) Biochemistry 34, 16004-16012]. The present paper focuses on the unique affects of F144R. Redox potential and EPR spectral properties of the Qo site of F144R showed that arginine forms an ion pair with the head group of an anionic ubiquinone, tentatively suggested to be a ubihydroquinone anion (QH-) in the Qos domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗