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Background The human telomere contains tandem repeat of (TTAGG) capable of forming a higher order DNA structure known as G-quadruplex. Porphyrin molecules such as TMPyP4 bind and stabilize G-quadruplex structure. Methods Isothermal titration calorimetry (ITC), circular dichroism (CD), and mass spectroscopy (ESI/MS), were used to investigate the interactions between TMPyP4 and the Co(III), Ni(II), Cu(II), and Zn(II) complexes of TMPyP4 (e.g. Co(III)-TMPyP4) and a model human telomere G-quadruplex (hTel22) at or near physiologic ionic strength ([Na+] or [K+] ≈ 0.15 M). Results The apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 all formed complexes having a saturation stoichiometry of 4:1, moles of ligand per mole of DNA. Binding of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 is described by a “four-independent-sites model”. The two highest-affinity sites exhibit a K in the range of 108 to 1010 M− 1 with the two lower-affinity sites exhibiting a K in the range of 104 to 105 M− 1. Binding of Co(III)-TMPyP4, and Zn(II)-TMPyP4, is best described by a “two-independent-sites model” in which only the end-stacking binding mode is observed with a K in the range of 104 to 105 M− 1. Conclusions In the case of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4, the thermodynamic signatures for the two binding modes are consistent with an “end stacking” mechanism for the higher affinity binding mode and an “intercalation” mechanism for the lower affinity binding mode. In the case of Co(III)-TMPyP4 and Zn(II)-TMPyP4, both the lower affinity for the “end-stacking” mode and the loss of the intercalative mode for forming the 2:1 complexes with hTel22 are attributed to the preferred metal coordination geometry and the presence of axial ligands. General significance The preferred coordination geometry around the metal center strongly influences the energetics of the interactions between the metallated-TMPyP4 and the model human telomeric G-quadruplex. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.

Background Extradiol dioxygenases are a family of nonheme iron (and sometimes manganese) enzymes that catalyze an O2-dependent ring-opening reaction in a biodegradation pathway of aromatic compounds. Here we characterize the thermodynamics of two substrates binding in homoprotocatechuate 2,3-dioxygenase (HPCD) prior to the O2 activation step. Methods This study uses microcalorimetry under an inert atmosphere to measure thermodynamic parameters associated with catechol binding to nonheme metal centers in HPCD. Several stopped-flow rapid mixing experiments were used to support the calorimetry experiments. Results The equilibria constant for 4-nitrocatechol and homoprotocatechuate binding to the iron(II) and manganese(II) forms of HPCD range from 2 × 104 to 1 × 106, suggesting there are distinctive differences in how the enzyme–substrate complexes are stabilized. Further experiments in multiple buffers allowed us to correct the experimental ΔH for substrate ionization and to fully derive the pH and buffer independent thermodynamic parameters for substrate binding to HPCD. Fewer protons are released from the iron(II) dependent processes than their manganese(II) counterparts. Conclusions Condition independent thermodynamic parameters for 4-nitrocatechol and homoprotocatechuate binding to HPCD are highly consistent with each other, suggesting these enzyme–substrate complexes are more similar than once thought, and the ionization state of metal coordinated waters may be playing a role in tuning redox potential and in governing reactivity. General significance Substrate binding to HPCD is a complex set of equilibria that includes ionization of substrate and water release, yet it is also the key step in O2 activation. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.