Biomedical subjects

P Leslie Dutton

Publications and source records attributed to P Leslie Dutton.

At least 19 recordsLinked to original sources

PubMed2006

Resilience of Rhodobacter sphaeroides cytochrome bc1 to heme c1 ligation changes.

Typically, c hemes are bound to the protein through two thioether bonds to cysteines and two axial ligands to the heme iron. In high-potential class I c-type cytochromes, these axial ligands are commonly His-Met. A change in this methionine axial ligand is often correlated with a dramatic drop in the heme redox potential and loss of function. Here we describe a bacterial cytochrome c with an unusual tolerance to the alternations in the heme ligation pattern. Substitution of the heme ligating methionine (M185) in cytochrome c1 of the Rhodobacter sphaeroides cytochrome bc1 complex with Lys and Leu lowers the redox midpoint potential but not enough to prevent physiologically competent electron transfer in these fully functional variants. Only when Met-185 is replaced with His is the drop in the redox potential sufficiently large to cause cytochrome bc1 electron transfer chain failure. Functional mutants preserve the structural integrity of the heme crevice: only the nonfunctional His variant allows carbon monoxide to bind to reduced heme, indicating a significant opening of the heme environment. This range of cytochrome c1 ligand mutants exposes both the relative resilience to sixth axial ligand change and the ultimate thermodynamic limits of operation of the cofactor chains in cytochrome bc1.

Electron Transport Complex III↗

PubMed2006

15N solid-state NMR provides a sensitive probe of oxidized flavin reactive sites.

Flavins are central to the reactivity of a wide variety of enzymes and electron transport proteins. There is great interest in understanding the basis for the different reactivities displayed by flavins in different protein contexts. We propose solid-state nuclear magnetic resonance (SS-NMR) as a tool for directly observing reactive positions of the flavin ring and thereby obtaining information on their frontier orbitals. We now report the SS-NMR signals of the redox-active nitrogens N1 and N5, as well as that of N3. The chemical shift tensor of N5 is over 720 ppm wide, in accordance with the predictions of theory and our calculations. The signal of N3 can be distinguished on the basis of coupling to 1H absent for N1 and N5, as well as the shift tensor span of only 170 ppm, consistent with N3's lower aromaticity and lack of a nonbonding lone pair. The isotropic shifts and spans of N5 and N1 reflect two opposite extremes of the chemical shift range for "pyridine-type" N's, consistent with their electrophilic and nucleophilic chemical reactivities, respectively. Upon flavin reduction, N5's chemical shift tensor contracts dramatically to a span of less than 110 ppm, and the isotropic chemical shift changes by approximately 300 ppm. Both are consistent with loss of N5's nonbonding lone pair and decreased aromaticity, and illustrate the responsiveness of the 15N chemical shift principal values to electronic structure. Thus. 15N chemical shift principal values promise to be valuable tools for understanding electronic differences that underlie variations in flavin reactivity, as well as the reactivities of other heterocyclic cofactors.

Binding Sites↗

PubMed2006

Nativelike structure in designed four alpha-helix bundles driven by buried polar interactions.

We show that a single internal polar interaction per helix is sufficient to engender structural specificity in that helix in helical bundle proteins. Furthermore, we use histidine-binding cofactors of different shapes which bind directly into the core, demonstrating that this structural specificity is not the result of a prescribed complimentary, "knobs in holes" core packing. We show that we can switch structural specificity of individual helices on and off by ligating cofactors, singly and in pairs, which bind either one or two histidine ligands. To our knowledge, this is the first demonstration of such extensive manipulation of protein structure by ligand binding, an important result of general interest to those working with self-assembled molecular systems. Finally, as these proteins were designed without the use of computational modeling, we not only demonstrate that designing a uniquely structured cofactor binding protein is not as difficult as is generally believed, we have determined why this is so: hydrophobic core complementarity, which is very difficult to design, is not necessary. Instead, a much simpler design process entails the creation of core polar interactions which themselves can drive conformational specificity.

Hydrogen Bonding↗

PubMed2006

Role of the PEWY glutamate in hydroquinone-quinone oxidation-reduction catalysis in the Qo Site of cytochrome bc1.

The glutamic acid residue of the conserved PEWY motif of the Q(o) site of cytochrome bc(1) is widely discussed as central to reversible Q(o) site catalysis of two-electron, two-proton hydroquinone-quinone oxidation-reduction. Extensive mutation of this glutamate (E295) to A, V, F, H, K, and Q in purple photosynthetic Rhodobacter capsulatus results in hydroquinone oxidation rates that are between 5 and 50-fold slower than that in the wild type. However, the mutants show little or no detectable effects on hydroquinone or quinone exchange and binding at the Q(o) site nor on subsequent Q(o) site-mediated redox equilibria in the c-chain and b-chain from pH 5-10. Lack of effects of mutations on the E(m)/pH plots rules out involvement of E295 in the strong electron-proton coupling evident in either the FeS center or heme b(L). These detailed equilibrium and kinetic analyses demonstrate that E295 is not irreplaceable in the Q(o) site catalytic mechanism. Rather, E295 and several other Q(o) site residues that can also be widely varied and still support hydroquinone oxidation illustrate the considerable resilience of Q(o) site activity to mutational change in Q(o) site environs. Residues and water molecules appear to cooperate in providing a physical and chemical environment supporting hydroquinone oxidation rates comparable to those seen in nonprotein aqueous environments at electrodes. We suggest that residues at the Q(o) site (and, possibly, other respiratory and photosynthetic quinone and oxygen binding sites) are a product of natural selection primarily acting not to lower catalytic barriers according to the traditional view of enzymatic catalysis but rather to develop specificity by raising barriers in defense of semiquinone loss or energy wasting short-circuit reactions.

Binding Sites↗

PubMed2006

Darwin at the molecular scale: selection and variance in electron tunnelling proteins including cytochrome c oxidase.

Biological electron transfer is designed to connect catalytic clusters by chains of redox cofactors. A review of the characterized natural redox proteins with a critical eye for molecular scale measurement of variation and selection related to physiological function shows no statistically significant differences in the protein medium lying between cofactors engaged in physiologically beneficial or detrimental electron transfer. Instead, control of electron tunnelling over long distances relies overwhelmingly on less than 14 A spacing between the cofactors in a chain. Near catalytic clusters, shorter distances (commonly less than 7 A) appear to be selected to generate tunnelling frequencies sufficiently high to scale the barriers of multi-electron, bond-forming/-breaking catalysis at physiological rates. We illustrate this behaviour in a tunnelling network analysis of cytochrome c oxidase. In order to surmount the large, thermally activated, adiabatic barriers in the 5-10 kcal mol-1 range expected for H+ motion and O2 reduction at the binuclear centre of oxidase on the 10(3)-10(5) s-1 time-scale of respiration, electron access with a tunnelling frequency of 10(9) or 10(10) s-1 is required. This is provided by selecting closely placed redox centres, such as haem a (6.9 A) or tyrosine (4.9 A). A corollary is that more distantly placed redox centres, such as CuA, cannot rapidly scale the catalytic site barrier, but must send their electrons through more closely placed centres, avoiding direct short circuits that might circumvent proton pumping coupled to haems a to a3 electron transfer. The selection of distances and energetic barriers directs electron transfer from CuA to haem a rather than a3, without any need for delicate engineering of the protein medium to 'hard wire' electron transfer. Indeed, an examination of a large number of oxidoreductases provides no evidence of such naturally selected wiring of electron tunnelling pathways.

Catalysis↗

PubMed2006

Quantum catalysis in enzymes: beyond the transition state theory paradigm. A Discussion Meeting held at the Royal Society on 14 and 15 November 2005.

How do enzymes work? What is the physical basis of the phenomenal rate enhancements achieved by enzymes? Do we have a theoretical framework that accounts for observed catalytic rates? These are the foremost questions-with particular emphasis on tunnelling phenomena-debated at this Discussion Meeting by the leading practitioners in the field.

Biocatalysis↗

PubMed2006

Intelligent design: the de novo engineering of proteins with specified functions.

One of the principal successes of de novo protein design has been the creation of small, robust protein-cofactor complexes which can serve as simplified models, or maquettes, of more complicated multicofactor protein complexes commonly found in nature. Different maquettes, generated by us and others, recreate a variety of aspects, or functional elements, recognized as parts of natural enzyme function. The current challenge is to both expand the palette of functional elements and combine and/or integrate them in recreating familiar enzyme activities or generating novel catalysis in the simplest protein scaffolds.

Catalysis↗

PubMed2006

Electron tunneling chains of mitochondria.

The single, simple concept that natural selection adjusts distances between redox cofactors goes a long way towards encompassing natural electron transfer protein design. Distances are short or long as required to direct or insulate promiscuously tunneling single electrons. Along a chain, distances are usually 14 A or less. Shorter distances are needed to allow climbing of added energetic barriers at paired-electron catalytic centers in which substrate and the required number of cofactors form a compact cluster. When there is a short-circuit danger, distances between shorting centers are relatively long. Distances much longer than 14 A will support only very slow electron tunneling, but could act as high impedance signals useful in regulation. Tunneling simulations of the respiratory complexes provide clear illustrations of this simple engineering.

Animals↗

PubMed2006

Design of a minimal polypeptide unit for bacteriochlorophyll binding and self-assembly based on photosynthetic bacterial light-harvesting proteins.

We introduce LH1beta24, a minimal 24 amino acid polypeptide that binds and assembles bacteriochlorophylls (BChls) in micelles of octyl beta-glucoside (OG) into complexes with spectral properties that resemble those of B820, a universal intermediate in the assembly of native purple bacterial light-harvesting complexes (LHs). LH1beta24 was designed by a survey of sequences and crystal structures of bacterial LH proteins from different organisms combined with currently available information from in vitro reconstitution studies and genetically modified LHs in vivo. We took as a template for the design sphbeta31, a truncated 31 amino acid analogue of the native beta-apoprotein from the core LH complex of Rhodobacter sphaeroides. This peptide self-assembles with BChls to form B820 and, upon cooling and lowering OG concentration, forms red-shifted B850 spectral species that are considered analogous to native LH complexes. We find that LH1beta24 self-assembles with BChl in OG to form homodimeric B820-type subunits comprising two LH1beta24 and two BChl molecules per subunit. We demonstrate, by modeling the structure using the highly homologous structure of LH2 from Rhodospirillum molischianum, that it has the minimal size for BChl binding. Additionally, we have compared the self-assembly of sphbeta31 and LH1beta24 with BChls and discovered that the association enthalpies and entropies of both species are similar to those measured for native LH1 from Rhodospirillum rubrum. However, sphbeta31 readily aggregates into intermediate higher oligomeric species and further to form B850 species; moreover, the assembly process of these oligomers is not reversible, and they are apparently large nonspecific BChl-peptide coaggregates rather than well-defined nativelike LH complexes. Similar aggregates were observed during LH1beta24 assembly, but these were formed less readily and required lower temperatures than sphbeta31. In view of these results, we reevaluate previous in vitro reconstitution studies and propose alternative templates for new designs.

Amino Acid Sequence↗

PubMed2006

Design and engineering of photosynthetic light-harvesting and electron transfer using length, time, and energy scales.

Decades of research on the physical processes and chemical reaction-pathways in photosynthetic enzymes have resulted in an extensive database of kinetic information. Recently, this database has been augmented by a variety of high and medium resolution crystal structures of key photosynthetic enzymes that now include the two photosystems (PSI and PSII) of oxygenic photosynthetic organisms. Here, we examine the currently available structural and functional information from an engineer's point of view with the long-term goal of reproducing the key features of natural photosystems in de novo designed and custom-built molecular solar energy conversion devices. We find that the basic physics of the transfer processes, namely, the time constraints imposed by the rates of incoming photon flux and the various decay processes allow for a large degree of tolerance in the engineering parameters. Moreover, we find that the requirements to guarantee energy and electron transfer rates that yield high efficiency in natural photosystems are largely met by control of distance between chromophores and redox cofactors. Thus, for projected de novo designed constructions, the control of spatial organization of cofactor molecules within a dense array is initially given priority. Nevertheless, constructions accommodating dense arrays of different cofactors, some well within 1 nm from each other, still presents a significant challenge for protein design.

Biophysical Phenomena↗

PubMed2005

Tunneling in PSII.

With available high resolution structures of PSII and a collection of reported redox midpoint potentials for most of the cofactors, it is possible to compare the expected electron tunneling rates with experimental rates to determine which electron transfer reactions are likely to reflect simply engineered electron tunneling, and which are more sophisticated and associated with large product rearrangements or the making and breaking of bonds. Reliable reorganization energies are largely lacking in this photosystem compared to PSI and purple bacteria and contribute about an order of magnitude uncertainty in tunneling rate estimates. Nevertheless it seems clear that as in purple bacterial reaction centers and PSI, with the notable exception of the oxygen evolving center, the majority of electron transfers within PSII are electron-tunneling limited at room temperature. Tunneling simulations also suggest that the short circuit between pheophytin and the adjacent chlorophyll cation may be fast enough to challenge triplet decay as the principle means of charge recombination from Q(A)(-) at room temperature.

Electron Transport↗

PubMed2005

Design of amphiphilic protein maquettes: controlling assembly, membrane insertion, and cofactor interactions.

We have designed polypeptides combining selected lipophilic (LP) and hydrophilic (HP) sequences that assemble into amphiphilic (AP) alpha-helical bundles to reproduce key structure characteristics and functional elements of natural membrane proteins. The principal AP maquette (AP1) developed here joins 14 residues of a heme binding sequence from a structured diheme-four-alpha-helical bundle (HP1), with 24 residues of a membrane-spanning LP domain from the natural four-alpha-helical M2 channel of the influenza virus, through a flexible linking sequence (GGNG) to make a 42 amino acid peptide. The individual AP1 helices (without connecting loops) assemble in detergent into four-alpha-helical bundles as observed by analytical ultracentrifugation. The helices are oriented parallel as indicated by interactions typical of adjacent hemes. AP1 orients vectorially at nonpolar-polar interfaces and readily incorporates into phospholipid vesicles with >97% efficiency, although most probably without vectorial bias. Mono- and diheme-AP1 in membranes enhance functional elements well established in related HP analogues. These include strong redox charge coupling of heme with interior glutamates and internal electric field effects eliciting a remarkable 160 mV splitting of the redox potentials of adjacent hemes that leads to differential heme binding affinities. The AP maquette variants, AP2 and AP3, removed heme-ligating histidines from the HP domain and included heme-ligating histidines in LP domains by selecting the b(H) heme binding sequence from the membrane-spanning d-helix of respiratory cytochrome bc(1). These represent the first examples of AP maquettes with heme and bacteriochlorophyll binding sites located within the LP domains.

Binding Sites↗

PubMed2005

Design of amphiphilic protein maquettes: enhancing maquette functionality through binding of extremely hydrophobic cofactors to lipophilic domains.

We demonstrate coordination of the extremely hydrophobic 13(2)-OH-Ni-bacteriochlorophyll (Ni-BChl) to the lipophilic domain of a novel, designed amphiphilic protein maquette (AP3) dispersed in detergent micelles [Discher et al. (2005) Biochemistry 44, 12329-12343]. Sedimentation velocity and equilibrium experiments and steady-state absorption spectra indicate that Ni-BChl-AP3 is a four-helix bundle containing one Ni-BChl axially ligated by one or two histidines. The nature of the ligation was pursued with ultrafast visible spectroscopy. While it is well established that light excitation of axially ligated mono- and bisimidazole Ni-BChl in solution leads to rapid imidazole dissociation and nanosecond recombination, there is no evidence of axial ligand dissociation in the light-excited Ni-BChl-AP3. This indicates that Ni-BChl is confined within the AP3 protein, ligated to histidines with severely restricted mobility. Dissociation constants show that Ni-BChl binding to AP3 is considerably weaker than the nanomolar range usual for heme and hydrophilic (HP) maquettes; moreover, there is a tendency for the Ni-BChl-AP3 four-helix bundles to dimerize into eight-helix bundles. Nevertheless, the preparation of the Ni-BChl-AP3 four-alpha-helix maquettes, supported by time-resolved spectroscopic analysis of the nature of the ligation, provides a viable new approach to AP maquette designs that address the challenges involved in binding extremely hydrophobic cofactors.

Binding Sites↗

PubMed2005

De novo design of a redox-active minimal rubredoxin mimic.

Metal-binding sites in metalloproteins frequently occur at the interfaces of elements of secondary structure, which has enabled the retrostructural analysis of natural proteins and the de novo design of helical bundles that bind metal ion cofactors. However, the design of metalloproteins containing beta-structure is less well developed, despite the frequent occurrence of beta-conformations in natural metalloproteins. Here, we describe the design and construction of a beta-protein, RM1, that forms a stable, redox-active 4-Cys thiolate Fe(II/III) site analogous to the active site of rubredoxin. The protein folds into a beta-structure in the presence and absence of metal ions and binds Fe(II/III) to form a redox-active site that is stable to repeated cycles of oxidation and reduction, even in an aerobic environment.

Binding Sites↗

PubMed2005

Direct observation of redox-linked histidine protonation changes in the iron-sulfur protein of the cytochrome bc1 complex by ATR-FTIR spectroscopy.

The redox-linked protonation chemistry of the iron-sulfur protein (ISP) of the cytochrome bc(1) complex was studied by analysis of the pH dependencies of redox difference spectra using perfusion/electrochemically induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The ISP of Rhodobacter capsulatus within the intact cytochrome bc(1) complex was analyzed in a mutant form in which the midpoint potential of cytochrome c(1) was lower than that of the ISP. This was compared to a soluble domain of the ISP from the bovine bc(1) complex. Spectra of in situ bacterial and isolated bovine proteins differ markedly only in part of their amide I regions with the in situ protein having additional pH-dependent component(s). Apart from this, both in situ and isolated proteins exhibited the same pH-dependent IR features in reduced minus oxidized difference spectra. Specifically, at high pH, a strong H/D insensitive negative band appeared at 1447/1450 cm(-)(1), together with a peak at 1310 cm(-)(1), the change of a 1267/1255 cm(-)(1) peak/trough into a simple 1266 cm(-)(1) peak, and a trough at 1107 cm(-)(1). Comparison with spectra of model materials indicates that all four signals arise from an imidazolate to imidazole transition of histidine, hence providing the first direct demonstration that histidine is the redox-linked protonation site of the ISP. The 1450 cm(-)(1) band can be assigned to a ring stretch that is unique to the imidazolate form of histidine. It is relatively sharp, has a high extinction coefficient, and provides a novel marker band for the detection of imidazolate intermediates in enzymatic mechanisms generally.

Amino Acid Substitution↗

PubMed2005

Fixing the Q cycle.

Mitchell's key insight that all bioenergetic membranes run on the conversion of redox energy into transmembrane electrical and proton gradients took the form 30 years ago of a working model of the Q cycle of cytochrome bc1, which operates reversibly on coupled electron and proton transfers of quinone at two binding sites on opposite membrane faces. His remarkable model still stands today, but he had no structural information to provide understanding into how dangerous short-circuit redox reactions were avoided. Now, it is clear that the Q cycle must be fixed with a special mechanism that avoids semiquinone-mediated short circuits. Either the redox states of the quinone electron-transfer partners double-gate the semiquinone-intermediate stability, or semiquinone is avoided altogether in concerted double-electron transfer.

Electron Transport↗

PubMed2005

Amphiphilic four-helix bundle peptides designed for light-induced electron transfer across a soft interface.

A family of four-helix bundle peptides were designed to be amphiphilic, possessing distinct hydrophilic and hydrophobic domains along the length of the bundle's exterior. This facilitates their vectorial insertion across a soft interface between polar and nonpolar media. Their design also now provides for selective incorporation of electron donor and acceptor cofactors within each domain. This allows translation of the designed intramolecular electron transfer along the bundle axis into a macroscopic charge separation across the interface.

Amino Acid Sequence↗

PubMed2004

ATR-FTIR spectroscopy studies of iron-sulfur protein and cytochrome c1 in the Rhodobacter capsulatus cytochrome bc1 complex.

Redox transitions in the Rhodobacter capsulatus cytochrome bc(1) complex were investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy combined with synchronous visible spectroscopy, in both the wild type and a cytochrome c(1) point mutant, M183K, in which the midpoint potential of heme was lowered from the wild-type value of 320 mV to 60 mV. Overall redox difference spectra of the wild type and M183K mutant were essentially identical, indicating that the mutation did not cause any major structural perturbation. Spectra were compared with data on the bovine bc(1) complex, and tentative assignments of several bands could be made by comparison with available data on model compounds and crystallographic structures. The bacterial spectra showed contributions from ubiquinone that were much larger than in the bovine enzyme, arising from additional bound and adventitious ubiquinone. The M183K mutant enabled selective reduction of the iron-sulfur protein which in turn allowed the IR redox difference spectra of ISP and cytochrome c(1) to be deconvoluted at high signal/noise ratios, and features of these spectra are interpreted in light of structural and mechanistic information.

Cytochromes c1↗