Applicability of fluorescence-based sensors to the determination of kinetic parameters for O2 in oxygenases.

Optical methods for O2 determination based on dynamic fluorescence quenching have been applied to measure oxygen uptake rates in cell culture and to determine intracellular oxygen levels. Here we demonstrate the applicability of fluorescence-based probes in determining kinetic parameters for O2 using as an example catalysis by a cofactor-independent oxygenase (DpgC). Fluorescence-based sensors provide a direct assessment of enzyme-catalyzed O2 consumption using commercially available, low-cost instrumentation that is easily customizable and, thus, constitutes a convenient alternative to the widely used Clark-type electrode, especially in cases where chemical interference is expected to be problematic.

Synthesis, characterization, and reactivity of iron(III) complexes supported by a trianionic ONO(3-) pincer ligand.

Synthetic and characterization results of a new family of Fe(III) compounds stabilized by a trianionic [CF3-ONO](3-) pincer-type ligand are reported. The ligand possesses three negatively charged donors constrained to the meridional positions that provide sufficient electron density to stabilize high-valent metal complexes. Using the redox-insulated [CF3-ONO](3-), pentacoordinated square-pyramidal {[CF3-ONO]FeCl2}{LiTHF2}2 (3), dimeric µ-DME{[CF3-ONO]FeDME}2 (4), trigonal bipyramidal [CF3-ONO]Fe(bpy) (5), and octahedral [CF3-ONO]Fe(bpy)H2O (5·H2O) complexes are synthesized. An interesting feature of the [CF3-ONO](3-) pincer-type ligand is its ability to coordinate the metal center in both the more common meridional positions or occupying a face of a trigonal bipyramidal complex. The molecular structure of 3 contains structural features similar to those of a rare square-planar high-spin Fe(II) complex, and the important role of the counterions in stabilizing a square-plane is emphasized. SQUID magnetometry measurements of 3 reveal its high-spin character, and cyclic voltammetry measurements indicate high oxidation state species are unstable. However, all compounds can be reduced, and in particular 5 displays a reversible reduction event at -2255 mV versus ferrocene (Fc(+)/Fc) that can be assigned to either the Fe(I)/Fe(0) couple or 2,2'-bipyridine reduction.

pH-Dependent conformational changes in proteins and their effect on experimental pK(a)s: the case of Nitrophorin 4.

The acid-base behavior of amino acids is an important subject of study due to their prominent role in enzyme catalysis, substrate binding and protein structure. Due to interactions with the protein environment, their pK(a)s can be shifted from their solution values and, if a protein has two stable conformations, it is possible for a residue to have different "microscopic", conformation-dependent pK(a) values. In those cases, interpretation of experimental measurements of the pK(a) is complicated by the coupling between pH, protonation state and protein conformation. We explored these issues using Nitrophorin 4 (NP4), a protein that releases NO in a pH sensitive manner. At pH 5.5 NP4 is in a closed conformation where NO is tightly bound, while at pH 7.5 Asp30 becomes deprotonated, causing the conformation to change to an open state from which NO can easily escape. Using constant pH molecular dynamics we found two distinct microscopic Asp30 pK(a)s: 8.5 in the closed structure and 4.3 in the open structure. Using a four-state model, we then related the obtained microscopic values to the experimentally observed "apparent" pK(a), obtaining a value of 6.5, in excellent agreement with experimental data. This value must be interpreted as the pH at which the closed to open population transition takes place. More generally, our results show that it is possible to relate microscopic structure dependent pKa values to experimentally observed ensemble dependent apparent pK(a)s and that the insight gained in the relatively simple case of NP4 can be useful in several more complex cases involving a pH dependent transition, of great biochemical interest.

Oxygen diffusion pathways in a cofactor-independent dioxygenase.

Molecular oxygen plays an important role in a wide variety of enzymatic reactions. Through recent research efforts combining computational and experimental methods a new view of O2 diffusion is emerging, where specific channels guide O2 to the active site. The focus of this work is DpgC, a cofactor-independent oxygenase. Molecular dynamics simulations, together with mutagenesis experiments and xenon-binding data, reveal that O2 reaches the active site of this enzyme using three main pathways and four different access points. These pathways connect a series of dynamic hydrophobic pockets, concentrating O2 at a specific face of the enzyme substrate. Extensive molecular dynamics simulations provide information about which pathways are more frequently used. This data is consistent with the results of kinetic measurements on mutants and is difficult to obtain using computational cavity-location methods. Taken together, our results reveal that although DpgC is rare in its ability of activating O2 in the absence of cofactors or metals, the way O2 reaches the active site is similar to that reported for other O2-using proteins: multiple access channels are available, and the architecture of the pathway network can provide regio- and stereoselectivity. Our results point to the existence of common themes in O2 access that are conserved among very different types of proteins.

Underlying thermodynamics of pH-dependent allostery.

Understanding the effects of coupling protein protonation and conformational states is critical to the development of drugs targeting pH sensors and to the rational engineering of pH switches. In this work, we address this issue by performing a comprehensive study of the pH-regulated switch from the closed to the open conformation in nitrophorin 4 (NP4) that determines its pH-dependent activity. Our calculations show that D30 is the only amino acid that has two significantly different pKas in the open and closed conformations, confirming its critical role in regulating pH-dependent behavior. In addition, we describe the free-energy landscape of the conformational change as a function of pH, obtaining accurate estimations of free-energy barriers and equilibrium constants using different methods. The underlying thermodynamic model of the switch workings suggests the possibility of tuning the observed pKa only through the conformational equilibria, keeping the same conformation-specific pKas, as evidenced by the proposed K125L mutant. Moreover, coupling between the protonation and conformational equilibria results in efficient regulation and pH-sensing around physiological pH values only for some combinations of protonation and conformational equilibrium constants, placing constraints on their possible values and leaving a narrow space for protein molecular evolution. The calculations and analysis presented here are of general applicability and provide a guide as to how more complex systems can be studied, offering insight into how pH-regulated allostery works of great value for designing drugs that target pH sensors and for rational engineering of pH switches beyond the common histidine trigger.