Metallocofactors are ubiquitous in nature, serving multiple purposes in proteins. These metallocofactors typically act as the site of catalysis or as an electron relay to move electrons within the protein, or within the cell, and are very energetically costly to manufacture. Yet, in nature it can appear that supernumerary, or ‘auxiliary’ cofactors are apparent, with no clear function. In this thesis, I address the question of what roles additional cofactors play, and why they are retained. The radical S-adenosylmethionine (AdoMet) enzyme superfamily has displayed great diversity in the cofactor requirements for its members. Some members of this family contain only the canonical [4Fe-4S] cluster, which reductively cleaves AdoMet to initiate chemistry, while others have additional [2Fe-2S] or [4Fe-4S] clusters. Even greater cofactor complexity is seen with the B12-dependent subclass, featuring a cobalamin-binding domain in addition to the canonical FeS cluster. The majority of this thesis has focused on using the technique of protein film electrochemistry (PFE) to study members of various subclasses of this superfamily: a dehydrogenase: BtrN, two methylthiotransferases: MiaB and RimO, as well as OxsB and TsrM, two B12-dependent enzymes. By evaluating the redox properties of members of different subclasses, we have been able to shed light on the redox properties of this superfamily, in general, and observed that the redox properties of auxiliary clusters can differ widely between subclasses (e.g. BtrN versus MiaB). PFE has also been used to evaluate five ferredoxins that are possible electron donors for MiaB from Thermotoga maritima. Additionally, bacterial cytochrome c peroxidases (bCCPs) are diheme enzymes catalyzing the detoxification of hydrogen peroxide; however, a novel subclass of bCCPs containing a third heme-binding motif has been identified in enteric pathogens. Protein film electrochemistry has been used to study the redox properties of Escherichia coli YhjA, a member of this subgroup. Further characterization of this novel bCCP was achieved with electron paramagnetic resonance, optical spectroscopy, and steady-state kinetics. Through characterizing YhjA and members of the AdoMet radical enzyme superfamily, we have shed light on the role these additional cofactors play in the mechanism and how these enzymes are tuned for their specific chemistries. / 2018-08-11T00:00:00Z

<p>In silico ligand docking is a versatile and common technique when predicting ligands and inhibitors for protein binding sites. The various docking programmes aim to calculate binding energies and to predict interactions, thus identifying potential ligands.The currently available programmes lack satisfying means by which to account for structural water molecules which can either mediate protein-ligand contacts or be displaced upon ligand binding. The present project aims to generate data to facilitate the global work of developing scoring functions in docking programmes to account for structural water molecules contribution to ligand binding to fill the said void. This is done by validating the performance of docking using a simple model system (cytochrome C peroxidase (CCP) W191G) containing four well ordered, deeply buried structural water molecules which are known to either interact with a ligand or to be displaced upon ligand binding.Known ligands were docked into eight (crystallographically determined) receptor set-ups comprising the receptor and no, one or two of the water molecules. The performance was validated by comparison of the binding modes of the docked ligands and the crystal structures, comparison of docking scores of the ligands in the different set-ups, enrichment of the ligands from a database of decoys and finally by predicting new ligands from the decoy database. In addition a high resolution crystal structure of CCP W191G in complex with 3-aminopyridine (3AP) was determined in order to resolve ambiguities in the binding mode of this ligand.</p>

Cardiac injury is increased following ischemia-reperfusion. Mitochondria are the “effector organelles” that are damaged during ischemia (ISC) when there is no blood flow. Resumption of metabolism by damaged mitochondria during reperfusion (REP) results in increased cell injury. Current therapeutic interventions to pre-condition and post-condition the heart during ISC are ineffective during certain conditions like aging and diabetes due to defects in the signaling cascades. In contrast, mitochondrial-based strategies are effective in protecting the heart during ISC-REP. Hence direct therapeutic targeting of dysfunctional mitochondria will provide the potential to bypass the upstream signaling defects and intervene directly upon the effector organelle. Novel mitochondrial-targeted therapy relies on understanding the sites in the electron transport chain (ETC) that are damaged by ISC and produce cell-injury during REP. This project identifies a novel pathological role of cytochrome c in depleting cardiolipin during ischemia after which the mitochondria are in a defective condition that leads to additional cell death during reperfusion. During ischemia oxidants from complex III oxidize cytochrome c, forming a peroxidase, which causes oxidative damage and depletion of cardiolipin. Depletion of cardiolipin disrupts normal physiology and augments cell death. Identification of the innovative pathobiology during ISC-REP recognizes a novel therapeutic target, cytochrome c peroxidase, which can be a focal point for new therapeutic interventions to decrease cardiac injury. In order to maintain homeostatis, living organisms have the methionine sulfoxide reductase system, which reduce both free and protein bound Met(O) back to methionine (Met) in the presence of thioredoxin. Oxidized Trx is inactive and unable to bind to ASK1 thereby activating ASK1 and causing cell death via p38/JNK pathways thereby contributing to the pathogenesis of myocardial ISC-REP injury. In this study we have shown that inhibition of ASK1 protects the heart during REP via the modulation of mitochondria that sustained damage during ISC. The mitochondrial-based mechanism of cardioprotection with ASK1 inhibition enhanced the functional integrity of the inner mitochondrial membrane retaining cytochrome c thereby decreasing cell death. This therapeutic intervention is a key step to achieve the ultimate goal to improve clinical outcomes in patients that suffer an acute myocardial infarction.

In silico ligand docking is a versatile and common technique when predicting ligands and inhibitors for protein binding sites. The various docking programmes aim to calculate binding energies and to predict interactions, thus identifying potential ligands.The currently available programmes lack satisfying means by which to account for structural water molecules which can either mediate protein-ligand contacts or be displaced upon ligand binding. The present project aims to generate data to facilitate the global work of developing scoring functions in docking programmes to account for structural water molecules contribution to ligand binding to fill the said void. This is done by validating the performance of docking using a simple model system (cytochrome C peroxidase (CCP) W191G) containing four well ordered, deeply buried structural water molecules which are known to either interact with a ligand or to be displaced upon ligand binding.Known ligands were docked into eight (crystallographically determined) receptor set-ups comprising the receptor and no, one or two of the water molecules. The performance was validated by comparison of the binding modes of the docked ligands and the crystal structures, comparison of docking scores of the ligands in the different set-ups, enrichment of the ligands from a database of decoys and finally by predicting new ligands from the decoy database. In addition a high resolution crystal structure of CCP W191G in complex with 3-aminopyridine (3AP) was determined in order to resolve ambiguities in the binding mode of this ligand.

2024 / The di-heme bacterial cytochrome c peroxidase (BCcP) superfamily can be divided into two groups based on biological functions. Canonical BCcPs reduce hydrogen peroxide to water by taking electrons from their redox partner - cytochrome c. In contrast, MauG and its orthologs are involved in protein post-translational modifications using the bis-Fe (IV) species. One of the key factors that is highly corelated with the diversity of biological functions of BCcPs is the distal heme ligand on the electron transfer heme (E heme), demonstrating the significant structure-function relationships of BCcPs. Using the sequence similarity network coupled with genome neighborhood analysis, a new group of BCcPs has been discovered from marine bacteria. The sequence analysis indicates the lack of distal ligand on E heme. In addition, the conserved genome context consisted of PhnDCE phosphonate transporter and manganese dependent transcriptional repressor MntR suggests potential functions in phosphorus metabolism under metal dependent regulation, which is beyond the current understanding of the functions of BCcP enzymes. The biochemical and biophysical characterization of RolA, the newly discovered BCcP from Roseovarius lutimaris, reveals that it possesses no peroxidase activity but reacts with O2 when fully reduced, setting it apart from all known BCcPs. In order to investigate the biological function of RolA, genetic manipulation tools have been developed in R. lutimaris. Three deletion mutants including R. lutimaris ∆rolA, ∆mntR and ∆rolA-∆mntR have been generated. The qPCR analysis of R. lutimaris WT vs ∆mntR has shown up-regulation of rolA upon mntR deletion, confirming the transcription repression of RolA by MntR. Native RolA has been tagged and over-expressed in R. lutimaris. The UV-vis and EPR spectra have demonstrated that the heme environment of native RolA is different from known BCcPs. Like recombinant RolA, native RolA has no peroxidase activity and reacts with O2 under fully reduced state. Preliminary studies using R. lutimaris ∆rolA and ∆rolA-∆mntR have shown utilization of phosphonates as sole phosphorus source. Bioinformatics analysis of PhnD from R. lutimaris suggests RolA might be related with hypophosphite transformation. The investigation of biological function of RolA using growth assay and in vitro enzymatic activity assay is ongoing. The identification of RolA expands the knowledge and diversity of the BCcP superfamily. / 2027-02-12T00:00:00Z