STM Studies of Synthetic Peptide Monolayers

We have used scanning probe microscopy to investigate self-assembled monolayers of chemically synthesized peptides. We find that the peptides form a dense uniform monolayer, above which is found a sparse additional layer. Using scanning tunneling microscopy, submolecular resolution can be obtained, revealing the alpha helices which constitute the peptide. The nature of the images is not significantly affected by the incorporation of redox cofactors (hemes) in the peptides.Comment: 4 pages, 3 figures (4 gifs); to appear in the Proceedings of the XIIth Int. Winterschool on Electronic Properties of Novel Materials "Molecular Nanostructures", Kirchberg/Tyrol, Febr. 199

Substitutions at position 146 of cytochrome b affect drastically the properties of heme b(L) and the Q(o) site of Rhodobacter capsulatus cytochrome bc\u3csub\u3e1\u3c/sub\u3e 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 (b(H) and b(L)) 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 (Q(o)) site and heme b(L) 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 (E(m,7)) of the high (b(H)) and low (b(L)) 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 Q(o) site of the mutant enzymes were unoccupied. Moreover, the g(z) signal of heme b(L), but not that of heme b(H), 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 b(L) 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 b(L), alters substrate accessibility and binding to the Q(o) site, and renders the bc1 complex inactive

Electrochemistry of ubiquinones Menaquinones and plastoquinones in aprotic solvents

AbstractFirst and second half-wave reduction potentials of a series of 1,4-benzo- and 1,4-naphtho-quinones related to the naturally occurring ubiquinones, plastoquinones and menaquinones are correlated with substituent effects. Notably, E12 of 2,3-dimethoxy-1,4-benzoquinone is positive of the values for the 2,5- and 2,6-dimethoxy isomers, and of the value for methoxy-1,4-benzoquinone. This phenomenon is attributed to steric inhibition of resonance when two methoxy groups occupy adjacent positions, and the significance of this orientation in the ubiquinone series is highlighted

Distance metrics for heme protein electron tunneling

AbstractThere is no doubt that distance is the principal parameter that sets the order of magnitude for electron-tunneling rates in proteins. However, there continue to be varying ways to measure electron-tunneling distances in proteins. This distance uncertainty blurs the issue of whether the intervening protein medium has been naturally selected to speed or slow any particular electron-tunneling reaction. For redox cofactors lacking metals, an edge of the cofactor can be defined that approximates the extent in space that includes most of the wavefunction associated with its tunneling electron. Beyond this edge, the wavefunction tails off much more dramatically in space. The conjugated porphyrin ring seems a reasonable edge for the metal-free pheophytins and bacteriopheophytins of photosynthesis. For a metal containing redox cofactor such as heme, an appropriate cofactor edge is more ambiguous. Electron-tunneling distance may be measured from the conjugated heme macrocycle edge or from the metal, which can be up to 4.8 Å longer. In a typical protein medium, such a distance difference normally corresponds to a ~1000 fold decrease in tunneling rate. To address this ambiguity, we consider both natural heme protein electron transfer and light-activated electron transfer in ruthenated heme proteins. We find that the edge of the conjugated heme macrocycle provides a reliable and useful tunneling distance definition consistent with other biological electron-tunneling reactions. Furthermore, with this distance metric, heme axially- and edge-oriented electron transfers appear similar and equally well described by a simple square barrier tunneling model. This is in contrast to recent reports for metal-to-metal metrics that require exceptionally poor donor/acceptor couplings to explain heme axially-oriented electron transfers

Constructing a man-made c-type cytochrome maquette in vivo:electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette

The successful use of man-made proteins to advance synthetic biology requires both the fabrication of functional artificial proteins in a living environment, and the ability of these proteins to interact productively with other proteins and substrates in that environment. Proteins made by the maquette method integrate sophisticated oxidoreductase function into evolutionarily naive, non-computationally designed protein constructs with sequences that are entirely unrelated to any natural protein. Nevertheless, we show here that we can efficiently interface with the natural cellular machinery that covalently incorporates heme into natural cytochromes c to produce in vivo an artificial c-type cytochrome maquette. Furthermore, this c-type cytochrome maquette is designed with a displaceable histidine heme ligand that opens to allow functional oxygen binding, the primary event in more sophisticated functions ranging from oxygen storage and transport to catalytic hydroxylation. To exploit the range of functions that comes from the freedom to bind a variety of redox cofactors within a single maquette framework, this c-type cytochrome maquette is designed with a second, non-heme C, tetrapyrrole binding site, enabling the construction of an elementary electron transport chain, and when the heme C iron is replaced with zinc to create a Zn porphyrin, a light-activatable artificial redox protein. The work we describe here represents a major advance in de novo protein design, offering a robust platform for new c-type heme based oxidoreductase designs and an equally important proof-of-principle that cofactor-equipped man-made proteins can be expressed in living cells, paving the way for constructing functionally useful man-made proteins in vivo