Thermodynamic Investigation into the Mechanisms of Proton-Coupled Electron Transfer Events in Heme Protein Maquettes

Abstract

To study the engineering requirements for proton pumping in energy-converting enzymes such as cytochrome c oxidase, the thermodynamics and mechanisms of proton-coupled electron transfer in two designed heme proteins are elucidated. Both heme protein maquettes chosen, heme b-[H10A24]2 and heme b-[delta7-His]2, are four-alpha-helix bundles that display pH-dependent heme midpoint potential modulations, or redox-Bohr effects. Detailed equilibrium binding studies of ferric and ferrous heme b with these maquettes allow the individual contributions of heme-protein association, iron-histidine ligation, and heme-protein electrostatics to be elucidated. These data demonstrate that the larger, less well-structured [H10A24]2 binds heme b in both oxidation states tighter than the smaller and more well-structured [Delta7-His]2 due to a stronger porphyrin-protein hydrophobic interaction. The 66 mV (1.5 kcal/mol) difference in their heme reduction potentials observed at pH 8.0 is due mostly to stabilization of ferrous heme in [H10A24]2 relative to [delta7-His]2. The data indicate that porphyrin-protein hydrophobic interactions and heme iron coordination are responsible for the Kd value of 37 nM for the heme b-[delta7-His]2 scaffold, while the affinity of heme b for [H10A24]2 is 20-fold tighter due to a combination of porphyrin-protein hydrophobic interactions, iron coordination, and electrostatic effects. The data also illustrate that the contribution of bis-His coordination to ferrous heme protein affinity is limited, <3.0 kcal/mol. The 1H+/1e- redox-Bohr effect of heme b-[H10A24]2 is due to the greater absolute stabilization of the ferric heme (4.1 kcal/mol) compared to the ferrous heme (1.4 kcal/mol) binding upon glutamic acid deprotonation, i.e., an electrostatic response mechanism. The 2H+/1e- redox-Bohr effect observed for heme b-[delta7-His]2 is due to histidine protonation and histidine dissociation of ferrous heme b upon reduction, i.e., a ligand loss mechanism. These results indicate that the contribution of porphyrin-protein hydrophobic interactions to heme affinity is critical to maintaining the heme bound in both oxidation states and eliciting an electrostatic response from these designed heme protein scaffolds.

Measurement of the Heme Affinity for Yeast Dap1p, and Its Importance in Cellular Function

“…As previously reported, [Δ7-His] 2 has a ferric K D value 160 pM and a ferrous heme K D value 40 nM at pH 8.0 (17). Upon titration of apo-[Δ7-His] 2 to a solution of as-isolated Dap1p, under ferric and ferrous conditions, K comp values are measured as 2.5 ± 1.0 and 35 ± 15, respectively, and with 1:1 heme to protein ratios in both cases.…”

“…At pH 8.0, [Δ7-His] 2 possesses a ferric heme K D value of 160 pM and a ferrous heme K D value 40 nM and is kinetically competent for heme transfer experiments (17). In the ferric heme state, apo-[Δ7-His] 2 was titrated into a solution of 15 µm Dap1p containing 2.8 µM bound heme which resulted in a shift in the Soret band maximum from 398 nm to 412 nm.…”

Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors

“…S3), which is the pH of the yeast cytosol (24). Given the relatively reducing cellular environment, E m Cytosol ∼ −320 mV vs. normal hydrogen electrode (NHE) (25), which is governed by the ratio of oxidized-to-reduced glutathione, and the reduction potential of aqueous heme, estimated to be between −50 mV and −220 mV vs. NHE (26,27), we propose that LH is biased toward the reduced state and assume herein that LH is 100% reduced. However, the actual fraction of LH that is reduced is dependent on its speciation and the degree to which it equilibrates with the glutathione redox buffer, both of which are unknown.…”

“…The genes for mKATE2 and CG6 used to construct HS1 were purchased from GENESCRIPT and codon-optimized for expression in E. coli and S. cerevisiae or humans. Standard published methods for protein expression and purification (18), heme binding studies (20,27), fluorimetry, UV/visible spectroscopy, microscopy, flow cytometry, immunoblotting (41), catalase activity (34), oxygen consumption (41), total heme analysis (42), and cell growth are outlined in SI Appendix, SI Materials and Methods.…”

Metal templated design of protein interfaces

“…Consequently, the supramolecular arrangement of MBPC-1 *Previous work by several groups has shown that various preexisting folds can be employed as stable scaffolds for hosting diverse metal coordination environments. Some of such engineered metalloproteins manifest properties characteristic of highly evolved natural systems, such as metal selectivity (30), redox tunability (31,32) as well as open coordination sites that allow for reactivity (33) and reversible small molecule binding (34). 1 These authors contributed equally to this work.…”