Oxidation-reduction and photophysical properties of isomeric forms of Safranin.

Safranine O is widely used in the bioenergetics community as an indicator dye to determine membrane potentials and as an electron transfer mediator in potentiometric titrations. Here we show that two different commercial preparations of Safranine O contain less than sixty percent by weight of the title compound, with the rest primarily consisting of two closely related safranine isomers. All three major isomer components were isolated using reverse phase HPLC and their structures determined using mass spectrometry and two-dimensional NMR. These Safranines have two-electron midpoint potentials ranging from -272 to -315 mV vs. SHE. We have also investigated the absorption and fluorescence spectra of the compounds and found that they display distinct spectral and photophysical properties. While this mixture may aid in Safranine O's utility as a mediator compound, membrane potential measurements must take this range of dye potentials into account.

Fluoride binding to characteristic heme-pocket centers: Insights into ligand stability.

The studies on the L. pectinata hemoglobins (HbI, HbII, and HbIII) are essential because of their biological roles in hydrogen sulfide transport and metabolism. Variation in the pH could also play a role in the transport of hydrogen sulfide by HbI and oxygen by HbII and HbIII, respectively. Here, fluoride binding was used to further understand the structural properties essential for the molecular mechanism of ligand stabilization as a function of pH. The data allowed us to gain insights into how the physiological roles of HbI, HbII, HbIII, adult hemoglobin (A-Hb), and horse heart myoglobin (Mb) have an impact on the heme-bound fluoride stabilization. In addition, analysis of the vibrational assignments of the met-cyano heme complexes shows varied strength interactions of the heme-bound ligand. The heme pocket composition properties differ between HbI (GlnE7 and PheB10) and HbII/HbIII (GlnE7 and TyrB10). Also, the structural GlnE7 stereo orientation changes between HbI and HbII/HbIII. In HbI, its carbonyl group orients towards the heme iron, while in HbII/HbIII, the amino group occupies this position. Therefore, in HbI, the interactions to the heme-bound fluoride ion, cyanide, and oxygen with GlnE7 via H-bonding are not probable. Still, the aromatic cage PheB10, PheCD1, and PheE11 may contribute to the observed stabilization. However, a robust H-bonding networking stabilizes HbII and HbIII, heme-bound fluoride, cyanide, and oxygen ligand with the OH and NH2 groups of TyrB10 and GlnE7, respectively. At the same time, A-Hb and Mb have moderate but similar ligand interactions controlled by their respective distal E7 histidine.

Designing Light-Activated Charge-Separating Proteins with a Naphthoquinone Amino Acid.

The first principles design of manmade redox-protein maquettes is used to clarify the physical/chemical engineering supporting the mechanisms of natural enzymes with a view to recapitulate and surpass natural performance. Herein, we use intein-based protein semisynthesis to pair a synthetic naphthoquinone amino acid (Naq) with histidine-ligated photoactive metal-tetrapyrrole cofactors, creating a 100 µs photochemical charge separation unit akin to photosynthetic reaction centers. By using propargyl groups to protect the redox-active para-quinone during synthesis and assembly while permitting selective activation, we gain the ability to employ the quinone amino acid redox cofactor with the full set of natural amino acids in protein design. Direct anchoring of quinone to the protein backbone permits secure and adaptable control of intraprotein electron-tunneling distances and rates.

Evaluation of heme peripheral group interactions in extremely low-dielectric constant media and their contributions to the heme reduction potential.

In this study, we measured the contributions of the ionization of the heme propionates to the reduction potentials of heme b and heme a (bis)N-methylimidazole complexes in various low-dielectric constant conditions. Additionally, we measured the effects of H-bond to the heme a formyl group on the reduction potential of the heme. The performed electrochemical measurements show that ionization of the heme propionates lead to the largest redox change in dichloromethane with no electrolyte. The measured reduction potential changes for heme b and heme a were -55 and -47 mV (±10 mV) per ionized propionate, respectively. For heme a, the study demonstrates how the dielectric constant of the medium is important in the magnification of the ΔpKa upon redox-linked ionization of the heme propionates and their roles in the proton pump of cytochrome c oxidase.

Electrochemical determination of heme-linked pKa values and the importance of using fluoride binding in heme proteins.

The ultraviolet-visible (UV-vis) spectroelectrochemical measurements of heme proteins in the presence of a heme-bound fluoride ion can be used as a probe for heme-linked ionizations of acid-base groups in the heme pocket. A detailed study of the pH dependence of the midpoint potential of skeletal horse myoglobin (Mb) with a heme-bound fluoride ion (Mb-F) reveals how protonation of the distal histidine (H64) changes the redox properties of the protein with a determined pKa of 5.3. In addition, fluoride binding in myoglobin provides a stabilization of -1.9 kcal/mol of the ferric Mb-F relative to ferric Mb without fluoride.

A three-dimensional printed cell for rapid, low-volume spectroelectrochemistry.

We have used three-dimensional (3D) printing technology to create an inexpensive spectroelectrochemical cell insert that fits inside a standard cuvette and can be used with any transmission spectrometer. The cell positions the working, counter, and reference electrodes and has an interior volume of approximately 200 µl while simultaneously providing a full 1-cm path length for spectroscopic measurements. This method reduces the time required to perform a potentiometric titration on a molecule compared with standard chemical titration methods and achieves complete electrolysis of protein samples within minutes. Thus, the device combines the best aspects of thin-layer cells and standard potentiometry.

Electrochemical and structural coupling of the naphthoquinone amino acid.

As a prelude to engineering artificial energy conversion proteins emulating biology, we examine the inclusion of a synthetic naphthoquinone amino acid in a characterized host-guest protein and determine the effects of its quinone and hydroquinone forms on the helix-coil distribution.

Manipulating Reduction Potentials in an Artificial Safranin Cofactor.

Safranines hold great promise as artificial flavin-like electron transfer cofactors with tunable properties. We report the design and chemical synthesis of the p-methoxy derivative of safranine O using a new synthetic route based on the Ulmann condensation. Spectroelectrochemical comparison of the purified parent safranine and this derivative demonstrates that the modification increases its two-electron reduction potential by 125 mV, or 5.75 kcal/mol. This modification also causes redshifts in the absorbance and fluorescence spectra of the cofactor, suggesting that it may find future utility in arrayed sensor applications.

Reversible proton coupled electron transfer in a peptide-incorporated naphthoquinone amino acid.

The synthesis of a naphthoquinone amino acid and its electrochemical characterization in a peptide is presented.

Hydrogen bond-free flavin redox properties: managing flavins in extreme aprotic solvents.

We report a simple, single step reaction that transforms riboflavin, which is insoluble in benzene, to tetraphenylacetyl riboflavin (TPARF), which is soluble in this solvent to over 250 mM. Electrochemical analysis of TPARF both alone and in complexes with two benzene-soluble flavin receptors: dibenzylamidopyridine (DBAP) and monobenzylamidopyridine (MBAP), prove that this model system behaves similarly to other previously studied flavin model systems which were soluble only in more polar solvents such as dichloromethane. Binding titrations in both benzene and dichloromethane show that the association constants of TPARF with its ligands are over an order of magnitude higher in benzene than dichloromethane, a consequence of the fact that benzene does not compete for H-bonds, but dichloromethane does. The alteration induced in the visible spectrum of TPARF in benzene upon the addition of DBAP is very similar to the difference produced by transfer to dichloromethane, further indicating that the flavin head group engages in a similar degree of hydrogen bonding with dichloromethane as with its ligands. This work underlines the importance of using a truly nonpolar solvent for the analysis of the effects of hydrogen bonding on the energetics of any biomimetic host-guest model system.

A Flavin Analogue with Improved Solubility in Organic Solvents.

We report the synthesis and initial electrochemical characterization of a benzene-soluble flavin analogue: N(10)-2,2-dibenzylethyl-7,8-dimethylisoalloxazine (DBF, 1). This analogue, which has an unmodified flavin headgroup, is intended for use in the spectroscopic examination of the electronic effects of flavin hydrogen bonding in simple model systems in aprotic, non-hydrogen bonding solvents. With future spectroscopic studies in mind, we have developed a synthetic route which allows the incorporation of isotopic labels using inexpensive starting materials.