Charge recombination rate constants vary no more than 3-fold for interprotein ET in the Zn-substituted wild type (WT) cytochrome c peroxidase (CcP):cytochrome c (Cc) complex and in complexes with four mutants of the Cc protein (i.e., F82S, F82W, F82Y, and F82I), despite large differences in the ET distance. Theoretical analysis indicates that charge recombination for all complexes involves a combination of tunneling and hopping via Trp191. For three of the five structures (WT and F82S(W)), the protein favors hopping more than that in the other two structures that have longer heme → ZnP distances (F82Y(I)). Experimentally observed biexponential ET kinetics is explained by the complex locking in alternative coupling pathways, where the acceptor hole state is either primarily localized on ZnP (slow phase) or on Trp191 (fast phase). The large conformational differences between the CcP:Cc interface for the F82Y(I) mutants compared to that the WT and F82S(W) complexes are predicted to change the reorganization energies for the CcP:Cc ET reactions because of changes in solvent exposure and interprotein ET distances. Since the recombination reaction is likely to occur in the inverted Marcus regime, an increased reorganization energy compensates the decreased role for hopping recombination (and the longer transfer distance) in the F82Y(I) mutants. Taken together, coupling pathway and reorganization energy effects for the five protein complexes explain the observed insensitivity of recombination kinetics to donor-acceptor distance and docking pose and also reveals how hopping through aromatic residues can accelerate long-range ET.
Abstract Background Adults with sickle cell disease (SCD) suffer early mortality and high morbidity. Many are not affiliated with SCD centers, defined as no ambulatory visit with a SCD specialist in 2 years. Negative social determinants of health (SDOH) can impair access to care. Hypothesis: Negative SDOH are more likely to be experienced by unaffiliated adults than adults who regularly receive expert SCD care. Methods Cross-sectional analysis of the SCD Implementation Consortium (SCDIC) Registry, a convenience sample at 8 academic SCD centers in 2017–2019. A Distressed Communities Index (DCI) score was assigned to each registry member’s zip code. Insurance status and other barriers to care were self-reported. Most patients were enrolled in the clinic or hospital setting. Results The SCDIC Registry enrolled 288 Unaffiliated and 2110 Affiliated SCD patients, ages 15-45y. The highest DCI quintile accounted for 39% of both Unaffiliated and Affiliated patients. Lack of health insurance was reported by 19% of Unaffiliated versus 7% of Affiliated patients. The most frequently selected barriers to care for both groups were “previous bad experience with the healthcare system” (40%) and “Worry about Cost” (17%). SCD co-morbidities had no straightforward trend of association with Unaffiliated status. The 8 sites’ results varied. Conclusion The DCI economic measure of SDOH was not associated with Unaffiliated status of patients recruited in the health care delivery setting. SCDIC Registrants reside in more distressed communities than other Americans. Other SDOH themes of affordability and negative experiences might contribute to Unaffiliated status. Recruiting Unaffiliated SCD patients to care might benefit from systems adopting value-based patient-centered solutions.
We report rapid photoinitiated intracomplex electron transfer (ET) within a “charge-disproportionated” myoglobin (Mb) dimer with greatly enhanced affinity. Two mutually supportive Brownian Dynamics (BD) interface redesign strategies, one a new “heme-filtering” approach, were employed to “break the symmetry” of a Mb homodimer by pairing Mb constructs with complementary highly positive and highly negative net surface charges, introduced through D/E → K and K → E mutations, respectively. BD simulations using a previously developed positive mutant, Mb(+6) = Mb(D44K/D60K/E85K), led to construction of the complementary negative mutant Mb(−6) = Mb(K45E, K63E, K95E). Simulations predict the pair will form a well-defined complex comprising a tight ensemble of conformations with nearly parallel hemes, at a metal–metal distance ∼18–19 Å. Upon expression and X-ray characterization of the partners, BD predictions were verified through ET photocycle measurements enabled by Zn-deuteroporphyrin substitution, forming the [ZnMb(−6), Fe3+Mb(+6)] complex. Triplet ET quenching shows charge disproportionation increases the binding constant by no less than ∼5 orders of magnitude relative to wild-type Mb values. All progress curves for charge separation (CS) and charge recombination (CR) are reproduced by a generalized kinetic model for the interprotein ET photocycle. The intracomplex ET rate constants for both CS and CR are increased by over 5 orders of magnitude, and their viscosity independence is indicative of true interprotein ET, rather than dynamic gating as seen in previous studies. The complex displays an unprecedented timecourse for CR of the CS intermediate I. After a laser flash, I forms through photoinduced CS, accumulates to a maximum concentration, then dies away through CR. However, before completely disappearing, I reappears without another flash and reaches a second maximum before disappearing completely.
Copper-substituted cytochrome c (CuCc) has been used as a structurally faithful, redoxinert inhibitor to probe the mechanism of electron transfer (ET) between Cc molecules and cytochrome c peroxidase (CcP). This inhibitor enhances photoinduced ET quenching of the triplet excited state of a zinc-substituted protein (ZnCcP or ZnCc) by its iron(III) partner (Fe3+Cc or Fe3+CcP). These results show that CcP and Cc form a ternary complex in which one Cc molecule binds tightly at a surface domain of CcP having low ET reactivity, whereas the second Cc molecule binds weakly to the 1:1 complex at a second domain with markedly greater (approximately 10(3)) reactivity. These results also rule out the possibility that Cc bound at the second domain cooperatively enhances ET to Cc at the first domain. The multiphasic kinetics observed for the photoproduced ET intermediate do not reflect electron self-exchange between two Cc molecules within the ternary complex.
We present the first steps in applying transverse relaxation-optimized spectroscopy (TROSY) techniques to the study of allosterism. Each β-chain of the hemoglobin (Hb) tetramer has 17 valine residues. We have 15 N-labeled the β-chain Val residues and detected 16 of the 17 1 H- 15 N correlation peaks for β-chain Val of the R state CO-Hb structure by using the TROSY technique. Sequence-specific assignments are suggested, based mainly on analysis of the 1 H pseudocontact-shift increments produced by oxidizing the diamagnetic R state HbCO to the paramagnetic R state fluoromet form. When possible, we support these assignments with sequential nuclear Overhauser effect (NOE) information obtained from a two-dimensional [ 1 H, 1 H]-NOESY-TROSY experiment (NOESY, NOE spectroscopy). We have induced further the R-T conformational change by adding the allosteric effector, inositol hexaphosphate, to the fluoromet-Hb sample. This change induces substantial increments in the 1 H and 15 N chemical shifts, and we discuss the implication of these findings in the context of the tentative sequence assignments. These preliminary results suggest that amide nitrogen and amide proton chemical shifts in a selectively labeled sample are site-specific probes for monitoring the allosteric response of the ensemble-averaged solution structure of Hb. More important, the chemical-shift dispersion obtained is adequate to permit a complete assignment of the backbone 15 N/ 13 C resonances upon nonselective labeling.
We report that when mixed-metal, [M, Fe] hemoglobin (Hb) hybrids, with Fe in one type of subunit and M = Zn or Mg in the other type, are embedded in clear poly(vinyl alcohol) (PVA) films, they exhibit inter-subunit electron transfer (ET) electron−nuclear tunneling down to cryogenic temperatures (5 K), making them the first protein system other than photosynthetic systems to exhibit such behavior. The rate constant for the (Fe2+Porphyrin) → (MPorphyrin)+ inter-subunit ET reaction shows a roughly temperature-invariant, quantum-tunneling regime from cryogenic temperatures (5 K) up to ca. 200 K. Some of the hybrids (depending on M and the Fe ligand) begin to show a strong increase in this ET rate constant at higher temperatures. This behavior is discussed here in terms of a recent heuristic description of ET in a glassy environment that accounts for the fact that slow solvent relaxation at low temperatures, and in particular upon cooling through a glassing transition, causes the reaction pathway to deviate from the path through the equilibrium transition state, and leads to the formation of nonequilibrium ET product states represented by points on the product surface other than that of the equilibrium product state. The analysis suggests that in regard to the dynamical modes of motion that control ET, the protein "medium" acts substantially like a frozen glass, even at room temperature. It further suggests that, although the protein acts largely as its own heat bath, the thermal characteristics of that heat bath can be modified by the external environment.
Some of us have previously reported the preparation of a dimeric form of the iron storage protein, bacterioferritin (Bfr), in which the native heme b is substituted with the photosensitizer, Zn(II)-protoporphyrin IX (ZnPP-Bfr dimer). We further showed that the ZnPP-Bfr dimer can serve as a photosensitizer for platinum-catalyzed H2 generation in aqueous solution without the usually added electron relay between photosensitizer and platinum (Clark, E. R., Inorg. Chem. 2017, 56, 4584−4593). We proposed reductive or oxidative quenching pathways involving the ZnPP anion radical (ZnPP•–) or the ZnPP cation radical, (ZnPP•+), respectively. The present report describes structural, photophysical, and photochemical properties of the ZnPP in the ZnPP-Bfr dimer. X-ray absorption spectroscopic studies at 10 K showed a mixture of five- and six-coordinated Zn centers with axial coordination by one long Zn–SγMet distance of ∼2.8 Å and ∼40% having an additional shorter Zn–S distance of ∼2.4 Å, in addition to the expected 4 nitrogen atom coordination from the porphyrin. The ZnPP in ZnPP-Bfr dimer was prone to photosensitized oxidation to ZnPP•+. The ZnPP•+ was rapidly reduced by ascorbic acid, which we previously determined was essential for photosensitized H2 production in this system. These results are consistent with an oxidative quenching pathway involving electron transfer from 3ZnPP* to platinum, which may be assisted by a flexible ZnPP axial coordination sphere. However, the low quantum yield for H2 production (∼1%) in this system could make reductive quenching difficult to detect, and can, therefore, not be completely ruled out. The ZnPP-Bfr dimer provides a simple but versatile framework for mechanistic assessment and optimization of porphyrin-photosensitized H2 generation without an electron relay between porphyrin and the platinum catalyst.
Hydroxyurea is an evidence-based disease-modifying therapy for sickle cell disease (SCD) but is underutilized. The Integration of Mobile Health into Sickle Cell Disease Care to Increase Hydroxyurea Utilization (meSH) multicenter study leveraged mHealth to deliver targeted interventions to patients and providers. SCD studies often underenroll; and recruitment strategies in the SCD population are not widely studied. Unanticipated events can negatively impact enrollment, making it important to study strategies that ensure adequate study accrual.
Intracomplex electron transfer (ET) occurs most often in intrinsically transient, low affinity complexes. As a result, the means by which adequate specificity and reactivity are obtained to support effective ET is still poorly understood. We report here on two such ET complexes: cytochrome b5 (cyt b5) in reaction with its physiological partners, myoglobin (Mb) and hemoglobin (Hb). These complexes obey the Dynamic Docking (DD) paradigm: a large ensemble of weakly bound protein−protein configurations contribute to binding in the rapid-exchange limit, but only a few are ET-active. We report the ionic-strength dependence of the second-order rate constant, k2, for photoinitiated ET from within all four combinations of heme-neutralized Zn deuteroporphyrin-substituted Mb/αHb undergoing ET with cyt b5, the four "corners" of a "heme-neutralization square". These experiments provide insights into the relative importance of both global and local electrostatic contributions to the binding of reactive configurations, which are too few to be observed directly. To interpret the variations of k2 arising from heme neutralization, we have developed a procedure by which comparisons of the ET rate constants for a heme-neutralization square permit us to decompose the free energy of reactive binding into individual local electrostatic contributions associated with interactions between (i) the propionates of the two hemes and (ii) the heme of each protein with the polypeptide of its partner. Most notably, we find the contribution from the repulsion between propionates of partner hemes to the reactive binding free energy to be surprisingly small, ΔG(Hb) ∼ +1 kcal/mol at ambient temperature, 18 mM ionic strength, and we speculate about possible causes of this observation. To confirm the fundamental assumption of these studies, that the structure of a heme-neutralized protein is unaltered either by substitution of Zn or by heme neutralization, we have obtained the X-ray structure of ZnMb prepared with the porphyrin dimethyl ester and find it to be nearly isostructural with the native protein.
