Statistical, DFT and Continuum Electrostatics Analysis of Histidine Ligated Hemes in the Non-redundant Heme Database in Model Complexes and in Cytochrome c Oxidase
Year of Dissertation:
2010
Heme plays an important role in biological oxidation--reduction chemistry. Important heme structural factors of are investigated here to understand how the redox potential is shifted when bound to proteins. A statistical analysis of a non-redundant heme database shows that the redox potentials of heme are significantly correlated with heme types and heme ligand types. The patterns of histidine ligand orientation, relative
Engineering cofactor and ligand binding in an artificial neuroglobin
Year of Dissertation:
2012
HP-7 is one artificial mutated oxygen transport protein, which operates via a mechanism akin to human neuroglobin and cytoglobin. This protein destabilizes one of two heme- ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which has a neutral hydrophobicity, slows gaseous ligand binding 22-fold, increases the affinity of the distal histidine ligand by a factor of thirteen, and decreases the binding affinity of carbon monoxide, a nonreactive oxygen analogue, three-fold. Paradoxically, it also decreases heme binding affinity by a factor of three in the reduced state and six in the oxidized state. Application of a two-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins. We have also examined the effects these mutations have on function. The Kd of the nonnreactive oxygen analogue carbon monoxide (CO) is only decreased three-fold, despite the large increase in distal histidine affinity engendered by the 22-fold decrease in the histidine ligand off-rate. This is a result of the four-fold increase in affinity for CO binding to the pentacoordinate state. Oxygen binds to HP7 with a Kd of 117 µM, while the mutant rapidly oxidizes when exposed to oxygen. EPR analysis of both ferric hemoproteins demonstrates that the mutation increases disorder at the heme binding site. NMR-detected deuterium exchange demonstrates that the mutation causes a large increase in water penetration into the protein core. The inability of the mutant protein may thus either be due to increased water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors.
Studying Heme Electrochemistry in Heme Proteins and Quinone Binding in Purple Bacterial Reaction Center Using Multi-Conformation Continuum Electrostatics
Year of Dissertation:
2011
Hemes are important redox cofactors. They are found in a variety of proteins and show a diversity of functions. The free energy of heme reduction in different proteins is found to vary over more than 18 kcal/mol. It is a challenge to determine how proteins manage to achieve this enormous range of Ems with a single type of redox cofactor. Proteins containing 141 unique hemes of a-, b- and c-type, with bis-His, His-Met and aquo-His ligation were calculated using Multi-Conformation Continuum Electrostatics (MCCE). The experimental Ems range over 800 mV from -350 mV in cytochrome c3 to 450 mV in cytochrome c peroxidase (vs. SHE). The quantitative analysis of the factors that modulate heme electrochemistry includes the interactions of the heme with its ligands, the solvent, the backbone, and sidechains. MCCE calculated Ems are in good agreement with measured values. The overview of heme proteins with known structures and Ems shows the lowest and highest potential hemes are c-type, while the b-type hemes are found in the middle Em range. In solution, bis-His ligation lowers the Em by ≈205 mV relative to hemes with His-Met ligands. The bis-His, aquo-His and His-Met ligated b-type hemes all cluster about Ems which are ≈200 mV more positive in protein than in water. In contrast, the low potential bis-His c-type hemes are shifted little from in solution, while the high potential His-Met c-type hemes are raised by ≈300 mV from solution. The analysis shows that no single type of interaction can be identified as the most important in setting heme electrochemistry in proteins. Therefore, different proteins use different aspects of their structures to modulate the in situ heme electrochemistry.
Conditions For Entanglement In Spin Systems And For Multipartite Entanglement
Year of Dissertation:
2012
This dissertation reports a series of studies of conditions for entanglement in spin systems and multipartite entanglement.