Electron transfer in ruthenium-modified recombinant cytochromes and myoglobins

Citation

Casimiro, Danilo Riguera (1994) Electron transfer in ruthenium-modified recombinant cytochromes and myoglobins. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/jh62-an41. https://resolver.caltech.edu/CaltechTHESIS:06152010-102901683

Abstract

The aim of the work described in Chapters 2-4 is to elucidate the role of the protein matrix in determining the distant electronic couplings for intramolecular electron- transfer (ET) reactions. The study focuses on two paradigmatic proteins - myoglobin, a non-ET molecule, and cytochrome c, a mitochondrial electron carrier. Site-directed mutants of human myoglobin were constructed each having a single surface histidine for ruthenium labeling. These histidines are at various distances from the heme center (9.5, His70; 12.7, His48; 15.5 Å, His83). Each mutant was derivatized with a pentaammineruthenium complex and the heme was substituted with a photoexcitable zinc mesoporphyrin. The rates of photoinduced and back ET between the histidine-bound Ru complex and the porphyrin were measured via a laser flash technique. The experimentally derived electronic couplings were found to decay exponentially with the intersite separation. Three site-directed mutants (Glu66His, Glu66His/Tyr67Phe, and Leu58His) of yeast iso-1-cytochrome c were constructed in order to examine whether intervening aromatic side chains affect the rates of intramolecular ET to a significant extent. The crystal structure of the wild-type protein indicates that the ET pathways involve aromatic side chains - a bridging tryptophan at position 59 (in the case of His58) or a tyrosine at 67 (for His66). Incorporation of the Tyr67Phe mutation in the His66 mutant modifies the ET path in a well defined manner. The rates of intramolecular ET from the ferroheme to ruthenium polypyridine complexes bound to the surface histidines were measured using a laser flash-quench technique. Comparison of the experimentally derived donor-acceptor couplings with those of other previously studied cytochromes does not indicate any significant rate enhancement in the presence of bridging aromatic side chains. Furthermore, the rates correlate reasonably well with the predictions of a σ-tunneling pathway model. The electronic couplings in these myoglobins and cytochromes were analyzed in the context of current theoretical models. The data on cytochrome c strongly support the presence of specific routes for ET to and from the heme. In contrast, myoglobin provides a homogeneous barrier for ET. A comprehensive analysis of the data on both proteins, however, suggests that the apparent square-barrier nature of the polypeptide matrix of myoglobin is symptomatic of the presence of several competing pathways that effectively cause the electronic couplings to scale with direct intersite separations. These results suggest that more complex factors such as stereoelectronic effects and multiple pathways can contribute significantly to the coupling through a structurally heterogeneous medium such as that of a protein. In Chapter 5, we have extended the use of a substitution-inert ruthenium polypyridine complex in introducing stabilizing intramolecular crosslinks in a protein. We found a dramatic increase in thermal stability of a yeast iso- 1 -cytochrome c mutant upon crosslinking two adjacent histidines (His39 and His58) on opposite strands of a p- sheet with bis(2,2'-bipyridine)ruthenium complex. The melting point of the Ru-modified cytochrome (72.8 °C) is 23.2 °C higher than that of the unmodified protein. Comparison with another Ru-modified di-histidine mutant suggests that the extent of the effect is largely dependent on the size of the loop generated by the crosslink. The technique should be readily applicable to stabilizing proteins with β-sheets. Chapter 6 describes a novel strategy for the expression and purification of a recombinant, nonfunctional axial-ligand mutant of iso-1-cytochrome c (Met80—>Ala) in S. cerevisiae. It involves coexpressing in the same plasmid(YEp213) the nonfunctional gene with a functional gene copy for complementation on a nonfermentable carbon medium. The functional gene encodes a product with an engineered metal-chelating site (His39 and His58) that enables efficient separation of the two isoforms by immobilized metal-affinity chromatography. The purified Met80—>Ala protein, which possesses a binding site for dioxygen and other exogenous ligands, was produced in quantities sufficient for extensive biophysical characterization. Absorption spectra of several derivatives of this mutant show striking similarities to those of the corresponding derivatives of horseradish peroxidase, myoglobin, and cytochrome P-450. The new method greatly expands the possible structural changes that can be incorporated into this paradigmatic protein.

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