Artificial enzymes have proven useful for catalyzing organic transformations in water with high product stereospecificity. Despite the high yields and selectivity exhibited by some of these catalysts, less is known about the differences in activity and catalytic lifetimes between various metal centers in these artificial enzymes. In this work, the small heme protein myoglobin (Mb) was modified through cofactor substitution and mutagenesis to develop a new set of catalysts for carbene transfer reactions. Due to the high activity of ruthenium porphyrins in organic solvents towards carbene transfer, it was hypothesized that a ruthenium porphyrin reconstituted into Mb (RuMb) would be a more active catalyst for carbene transfer reactions than Fe-containing (native) Mb. Indeed, the ruthenium porphyrin (RuMpIX) in water exhibits a higher yield for both the N-H insertion of aniline (48% vs. 27%) and the cyclopropanation of styrene (26% vs. 16%) compared to the similar iron porphyrin FePpIX found in native Mb (FeMb). RuMb and FeMb variants exhibit similar yields for the N-H insertion of aniline (30-60%) with slightly higher yields achieved by the more hydrophobic H64A and H64V mutants relative to wild-type and H64D Mb. In contrast, all RuMb variants exhibit low activity towards the cyclopropanation of styrene (0-4% yield) compared to FeMb variants (25-36% yield). My work demonstrates that this is due to the fact that RuMpIX and RuMb both exhibit short catalyst lifetimes (≤ 30 minutes), and that the reactive metallocarbene formed during catalysis inserts into both the porphyrin and protein scaffold (as evidenced by UV-vis and mass spectrometry studies). RuMpIX also exhibits a higher rate constant of product formation (kobs = 5.9 x 10-2 min-1) compared to FePplX (kobs = 2.9 x 10-2 min-1), but suffers a shorter catalytic lifetime than FePplX (30 min vs. 120 min). Therefore, the Ru=CR2 unit is quite reactive towards nucleophiles, but this high reactivity results in carbene insertion into the porphyrin and protein, leading to rapid catalyst decomposition. Efforts towards ;;taming” the Ru catalyst by using more electron-donating proximal ligands (in the H93G mutant of Mb) have not been successful so far. Future studies should focus on gaining a better understanding of the active site of RuMb and on the decomposition products after the addition of the carbene precursor. A crystal structure of RuMb would lend greater insight into the structure of the active site and the orientation of RuMpIX in the heme pocket. Modulating carbene insertion activity by adding carbene precursors with electron-donating groups could also result in lower activity towards porphyrin or protein insertion and therefore longer catalyst lifetimes. In addition, this work investigates the mechanism of diheme bacterial cytochrome c peroxidases (CcPs). There are two classes of CcPs: some enzymes are active in the diferric form, and other enzymes must be reduced by one electron before catalytic activity commences. Using several different spectroscopic methods, the oxidation and spin states of the heme cofactors in two different CcPs were studied. This work suggests that the low-potential, peroxidatic heme remains low-spin ferric even in the active form of each enzyme. The active heme does not reside in a statically five-coordinate form and activation prior to peroxide binding is likely a more dynamic process than previously hypothesized. This could have implications for how the heme cofactors in other diheme enzymes interact and how activity is induced in different peroxidases.
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Heme Protein Engineering and Mechanistic Investigations
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