Comparison of the Backbone Dynamics of Dehaloperoxidase-Hemoglobin Isoenzymes.

Dehaloperoxidase (DHP) is a multifunctional hemeprotein with a functional switch generally regulated by the chemical class of the substrate. Its two isoforms, DHP-A and DHP-B, differ by only five amino acids and have an almost identical protein fold. However, the catalytic efficiency of DHP-B for oxidation by a peroxidase mechanism ranges from 2- to 6-fold greater than that of DHP-A depending on the conditions. X-ray crystallography has shown that many substrates and ligands have nearly identical binding in the two isoenzymes, suggesting that the difference in catalytic efficiency could be due to differences in the conformational dynamics. We compared the backbone dynamics of the DHP isoenzymes at pH 7 through heteronuclear relaxation dynamics at 11.75, 16.45, and 19.97 T in combination with four 300 ns MD simulations. While the overall dynamics of the isoenzymes are similar, there are specific local differences in functional regions of each protein. In DHP-A, Phe35 undergoes a slow chemical exchange between two conformational states likely coupled to a swinging motion of Tyr34. Moreover, Asn37 undergoes fast chemical exchange in DHP-A. Given that Phe35 and Asn37 are adjacent to Tyr34 and Tyr38, it is possible that their dynamics modulate the formation and migration of the active tyrosyl radicals in DHP-A at pH 7. Another significant difference is that both distal and proximal histidines have a 15-18% smaller S2 value in DHP-B, thus their greater flexibility could account for the higher catalytic activity. The distal histidine grants substrate access to the distal pocket. The greater flexibility of the proximal histidine could also accelerate H2O2 activation at the heme Fe by increased coupling of an amino acid charge relay to stabilize the ferryl Fe(IV) oxidation state in a Poulos-Kraut "push-pull"-type peroxidase mechanism.

Creatine and creatinine quantified using nuclear magnetic resonance: A method validation study and clinical associations between circulating creatine and fatigue in kidney transplant recipients.

BACKGROUND: A potential contributor to fatigue in kidney transplant recipients (KTR) may be impaired creatine homeostasis. We developed and validated a high-throughput NMR assay allowing for simultaneous measurement of circulating creatine and creatinine, and determined plasma creatine and estimated intramuscular creatine concentrations in KTRs, delineated their determinants and explored their associations with self-reported fatigue. METHODS: An NMR assay was developed and validated for measurement of circulating creatinine and creatine concentrations. Plasma creatine and creatinine concentrations were measured in 618 KTR. Fatigue was assessed using the checklist individual strength. Associations of creatine parameters with fatigue was assessed using linear mixed effect models. RESULTS: The NMR-based assay had good sensitivity, precision and demonstrated linearity across a large range of values. Among KTR, the mean age was 56 ± 13 years, 62% were men and eGFR was 54 ± 18 ml/min/1.73 m2. Plasma creatine concentration was 27 [19-39] µmol/L. Estimated intramuscular creatine concentration was 27 ± 7 mmol/kg. Higher plasma creatine concentration and higher estimated intramuscular creatine concentration were independently associated with a lower total fatigue score and less motivation problems. CONCLUSION: An NMR method for measurement of circulating creatine and creatinine which offers the potential for accurate and efficient quantification was developed. The found associations suggest that improving creatine status may play a beneficial role in mitigating fatigue.

Electronic structure of covalently linked zinc bacteriochlorin molecular arrays: insights into molecular design for NIR light harvesting.

Pigment-based molecular arrays, especially those based on porphyrins, have been extensively studied as viable components of artificial light harvesting devices. Unlike porphyrins, bacteriochlorins absorb strongly in the NIR, yet little is known of the applicability of covalently linked bacteriochlorin-based arrays in this arena. To lay the foundation for future studies of excited state properties of such arrays, we present a systematic study of the ground state electronic structure of zinc bacteriochlorin (ZnBC) molecular arrays with various linkers and linker attachment sites (meso vs ß) employing density functional theory in combination with the energy-based fragmentation (EBF) method, and the EBF with molecular orbitals (EBF-MO) method. We find that the level of steric hindrance between the ZnBC and the linker is directly correlated with the amount of ground sate electronic interactions between the ZnBCs. Low steric hindrance between the ZnBC and the linker found in alkyne-linked arrays results in strongly interacting arrays that are characterized by a decrease in the HOMO-LUMO energy gaps, large orbital energy dispersion in the frontier region, and low ZnBC-linker rotational barriers. In contrast, sterically hindered linkers, such as aryl-based linkers, result in weakly interacting arrays characterized by increased orbital energy degeneracy in the frontier region and high ZnBC-linker rotational barriers. For all linkers studied, the level of steric hindrance decreases when the ZnBCs are linked at the ß position. Hence, ZnBC arrays that exhibit strong, weak, or intermediate ground-state electronic interactions can be realized by adjusting the level of steric hindrance with a judicious choice of the linker type and linker attachment site. Such tuning may be essential for design of light harvesting arrays with desired spectral properties.