Biology subjects

Borgstahl, G. E.

Publications and source records attributed to Borgstahl, G. E..

5 recordsLinked to original sources

bioRxiv2025

Mapping the Exit Route of Hydrogen Peroxide From the Manganese Superoxide Dismutase (MnSOD) Active Site.

Human mitochondrial manganese superoxide dismutase (MnSOD) converts superoxide (O [bullet]-) into hydrogen peroxide (H O ) and molecular oxygen (O ), serving as a key defense against oxidative damage. Despite extensive studies, the full structural characterization of H2O2-binding sites in MnSOD remains largely unexplored. Previous H2O2-soaked MnSOD structures have identified two distinct H2O2-binding sites: one directly ligated to the catalytic Mn (LIG position) and another at the active site gateway (PEO position) between second-shell residues Tyr34 and His30. In this study, a kinetically impaired Gln143Asn MnSOD variant is used to trap and explore additional H2O2-binding sites beyond the second-shell solvent gate. In the wild-type enzyme, Gln143 mediates proton transfers with the Mn-bound solvent (WAT1) to drive redox cycling of the metal, necessary for effective O [bullet]- dismutation. Substitution with Asn stalls catalysis because the increased distance from WAT1 disrupts critical proton-coupled electron transfer (PCET) events, and the redox cycling of the active site metal is impaired. This, in turn, stalls the electrostatic cycling of positive charge on the enzyme surface and enhances the likelihood of trapping transient H2O2-bound states in this variant. Results reveal several H2O2 molecules leading up to the active site, in addition to the canonical LIG and PEO positions. SynopsisA high-resolution X-ray structure of a Gln143Asn variant of manganese superoxide dismutase reveals multiple hydrogen peroxide binding sites beyond the canonical LIG and PEO binding positions within the active site. These findings expand the known landscape of product peroxide-bound states in MnSOD.

biochemistry↗

bioRxiv2024

The role of Tyr34 in proton-coupled electron transfer of human manganese superoxide dismutase

Human manganese superoxide dismutase (MnSOD) plays a crucial role in controlling levels of reactive oxygen species (ROS) by converting superoxide (O2*-) to molecular oxygen (O2) and hydrogen peroxide (H2O2) with proton-coupled electron transfers (PCETs). The reactivity of human MnSOD is determined by the state of a key catalytic residue, Tyr34, that becomes post-translationally inactivated by nitration in various diseases associated with mitochondrial dysfunction. We previously reported that Tyr34 has an unusual pKa due to its proximity to the Mn metal and undergoes cyclic deprotonation and protonation events to promote the electron transfers of MnSOD. To shed light on the role of Tyr34 MnSOD catalysis, we performed neutron diffraction, X-ray spectroscopy, and quantum chemistry calculations of Tyr34Phe MnSOD in various enzymatic states. The data identifies the contributions of Tyr34 in MnSOD activity that support mitochondrial function and presents a thorough characterization of how a single tyrosine modulates PCET catalysis.

biochemistry↗

bioRxiv2024

Revealing the atomic and electronic mechanism of human manganese superoxide dismutase product inhibition

Human manganese superoxide dismutase (MnSOD) is a crucial oxidoreductase that maintains the vitality of mitochondria by converting O2[bullet]- to O2 and H2O2 with proton-coupled electron transfers (PCETs). Since changes in mitochondrial H2O2 concentrations are capable of stimulating apoptotic signaling pathways, human MnSOD has evolutionarily gained the ability to be highly inhibited by its own product, H2O2. A separate set of PCETs is thought to regulate product inhibition, though mechanisms of PCETs are typically unknown due to difficulties in detecting the protonation states of specific residues that coincide with the electronic state of the redox center. To shed light on the underlying mechanism, we combined neutron diffraction and X-ray absorption spectroscopy of the product-bound, trivalent, and divalent states to reveal the all-atom structures and electronic configuration of the metal. The data identifies the product-inhibited complex for the first time and a PCET mechanism of inhibition is constructed.

biochemistry↗

bioRxiv2023

Design, synthesis, and evaluation of a mitoxantrone probe (MXP) for biological studies

Mitoxantrone (MX) is a robust chemotherapeutic with well-characterized applications in treating certain leukemias and advanced breast and prostate cancers. The canonical mechanism of action associated with MX is its ability to intercalate DNA and inhibit topoisomerase II, giving it the designation of a topoisomerase II poison. Years after FDA approval, investigations have unveiled novel protein-binding partners, such as methyl-CpG-binding domain protein (MBD2), PIM1 serine/threonine kinase, RAD52, and others that may contribute to the therapeutic profile of MX. Moreover, recent proteomic studies have revealed MXs ability to modulate protein expression, illuminating the complex cellular interactions of MX. Although mechanistically relevant, the differential expression across the proteome does not address the direct interaction with potential binding partners. Identification and characterization of these MX-binding cellular partners will provide the molecular basis for the alternate mechanisms that influence MXs cytotoxicity. Here, we describe the design and synthesis of a MX-biotin probe (MXP) and negative control (MXP-NC) that can be used to define MXs cellular targets and expand our understanding of the proteome-wide profile for MX. In proof of concept studies, we used MXP to successfully isolate a recently identified protein-binding partner of MX, RAD52, in a cell lysate pulldown with streptavidin beads and western blotting. Graphical abstract (Draft) O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY HighlightsO_LIAn 8-step synthesis was used to generate a biotinylated-mitoxantrone probe (MXP). C_LIO_LIA pulldown of MXP demonstrated selectivity for RAD52, but not Replication Protein A. C_LIO_LIWestern blot confirmed the identity of the isolated protein, RAD52. C_LI

biochemistry↗

bioRxiv2020

Direct detection of coupled proton and electron transfers in human manganese superoxide dismutase

Human manganese superoxide dismutase (MnSOD) is a critical oxidoreductase found in the mitochondrial matrix. Concerted proton and electron transfers (CPETs) are used by the enzyme to rid the mitochondria of O2 *-, a precursor to other harmful reactive oxygen and nitrogen species. The mechanisms of CPET-utilizing enzymes are typically unknown due to the difficulties in detecting the protonation states of specific residues and solvent molecules involved in catalysis while controlling the redox state of the enzyme. Here, neutron diffraction of redox-controlled MnSOD crystals revealed the all-atom structures of Mn3+SOD and Mn2+SOD delivering unique data on sites that change protonation state. A novel mechanism is proposed from the direct observation of glutamine deprotonation, the involvement of Tyr and His with altered pKas, and four unusual strong-short hydrogen bonds, including a low barrier hydrogen bond, that change with the oxidation state of the metal. Quantum calculations provide insight into the electronic modulation of the observed structures and the enzymatic mechanism.

biochemistry↗