Modification of Heme Proteins for Reactivity and Mechanistic Studies

Abstract

In the past decade, carbene transfer biocatalysis has evolved from basic scientific research to an area with vast potential for the development of new industrial processes in the pharmaceutical industry. In this work YfeX, naturally a peroxidase, is shown to have a great potential for the development of new carbene transferases. Intrinsic reactivity of wild-type (WT) YfeX is in many cases on par with the best Mb variants available, and this protein shows high stability against organic co-solvents, thereby enabling us to solubilize hydrophobic substrates to improve turnover. In the cyclopropanation of styrene, WT YfeX naturally generates the trans product with 87% selectivity for the (R,R) enantiomer. WT YfeX also catalyzes N-H insertion with aliphatic amines (benzylamine) in high yield. Most excitingly, YfeX can catalyze the Si—H insertion of dimethylphenylsilane with 11% yield, which is the highest yield for any WT protein observed so far, and QM/MM calculations reveal further details of the mechanism of the unusual Si—H insertion reaction.

To explore the steric and electrostatic effects of the second coordination sphere near the active site of YfeX, utilizing rational design, four YfeX variants (I230A, D143A, R232A, and S234A) were investigated for enhanced carbene transferase reactivity. It was shown that R232A (with 75% yield) and I230A (with 92% yield) variants have increased N-H insertion reactivity and are a good starting point to further improve YfeX reactivity. These studies demonstrate that YfeX and variants are great biocatalysts and motivate the development of novel YfeX carbene transferases.

The second part of my thesis focuses on the investigation of the electron storage and distribution properties within the pentaheme scaffold of Geobacter lovleyi cytochrome c nitrite reductase (NrfA or ccNiR), a newly discovered subclass of cytochrome c nitrite reductases. NrfA from G. lovleyi, has emerged as a model representative of bacteria that have an important role in the global nitrogen cycle via the dissimilatory nitrate reduction to ammonium (DNRA) pathway. Initially, a chemical reduction method was established to sequentially add electrons to the fully oxidized protein, which was then studied using UV-Vis and electron paramagnetic resonance (EPR) spectroscopy. Based on quantitative analysis and simulation of the EPR data, we demonstrate that Hemes 1, 3, and 4 are exchange coupled, and the EPR signals of all five hemes in fully oxidized NrfA could be identified for the first time. EPR-spectral simulations were used to elucidate the sequence of heme reduction and reveal that Hemes 5 and 4 are reduced first (before the active site Heme 1) and can serve the purpose of an electron storage unit within the protein, instead of merely serving as a wire to pass electrons into Heme 1. Additionally, to probe the role of the central Heme 3, a H108M NrfA variant was generated with a positively shifted reduction potential, making it the first heme to be reduced. This H108M variant has a significant impact on the distribution of electrons within the pentaheme scaffold and decreases the catalytic activity of the enzyme to 3% compared to WT NrfA. These studies demonstrate that the four bis-His hemes of NrfA are much more than just a wire that allows for electron transfer to the active site Heme 1.

Furthermore, this work elucidates fundamental information about the complicated mechanism of electron storage within Hemes 4 and 5 and the overall electron distribution in NrfA.