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Replacing the iron atom in Fe-porphyrin IX proteins with a noble-metal atom enables the creation of enzymes that catalyse reactions not catalysed by native Fe-enzymes or other metalloenzymes; this approach could be used to generate other artificial enzymes that could catalyse a wide range of abiological transformations. Changing the 'metallo' in metalloenzymes Naturally occurring metalloenzymes are promising alternatives to transition-metal catalysts and non-metal enzymes for the synthesis of chemicals and biologically active compounds, but they catalyse only a narrow range of reactions. One way of broadening that range is to replace the native catalytic metal with an abiological metal. John Hartwig and colleagues report the effect of substituting the iron atom in Fe-porphyrin IX (Fe-PIX) proteins. Myoglobin variants containing an Ir(Me) site catalyse the functionalization of C–H bonds to form C–C bonds and add carbenes to β-substituted vinylarenes and unactivated aliphatic α-olefins. Directed evolution of the Ir(Me)-myoglobin generates mutants that form either enantiomer of the products of C–H insertion and catalyse the enantio- and diastereoselective cyclopropanation of unactivated olefins. The rich chemistry of free metalloporphyrins and the ease of preparation and evolution of substituted haem proteins using the methods adopted here open the way to the creation of many artificial metalloenzymes. Enzymes that contain metal ions—that is, metalloenzymes—possess the reactivity of a transition metal centre and the potential of molecular evolution to modulate the reactivity and substrate-selectivity of the system 1 . By exploiting substrate promiscuity and protein engineering, the scope of reactions catalysed by native metalloenzymes has been expanded recently to include abiological transformations 2 , 3 . However, this strategy is limited by the inherent reactivity of metal centres in native metalloenzymes. To overcome this limitation, artificial metalloproteins have been created by incorporating complete, noble-metal complexes within proteins lacking native metal sites 1 , 4 , 5 . The interactions of the substrate with the protein in these systems are, however, distinct from those with the native protein because the metal complex occupies the substrate binding site. At the intersection of these approaches lies a third strategy, in which the native metal of a metalloenzyme is replaced with an abiological metal with reactivity different from that of the metal in a native protein 6 , 7 , 8 . This strategy could create artificial enzymes for abiological catalysis within the natural substrate binding site of an enzyme that can be subjected to directed evolution. Here we report the formal replacement of iron in Fe-porphyrin IX (Fe-PIX) proteins with abiological, noble metals to create enzymes that catalyse reactions not catalysed by native Fe-enzymes or other metalloenzymes 9 , 10 . In particular, we prepared modified myoglobins containing an Ir(Me) site that catalyse the functionalization of C–H bonds to form C–C bonds by carbene insertion and add carbenes to both β-substituted vinylarenes and unactivated aliphatic α-olefins. We conducted directed evolution of the Ir(Me)-myoglobin and generated mutants that form either enantiomer of the products of C–H insertion and catalyse the enantio- and diastereoselective cyclopropanation of unactivated olefins. The presented method of preparing artificial haem proteins containing abiological metal porphyrins sets the stage for the generation of artificial enzymes from innumerable combinations of PIX-protein scaffolds and unnatural metal cofactors to catalyse a wide range of abiological transformations.

Neuroglobin, which is a heme protein from the globin family that is predominantly expressed in nervous tissue, can promote a neuronal survivor. However, the molecular mechanisms underlying the neuroprotective function of Ngb remain poorly understood to this day. The interactions between neuroglobin and mitochondrial cytochrome c may serve as at least one of the mechanisms of neuroglobin-mediated neuroprotection. Interestingly, neuroglobin and cytochrome c possibly can interact with or without electron transfer both in the cytoplasm and within the mitochondria. This review provides a general picture of molecular interactions between neuroglobin and cytochrome c based on the recent experimental and computational work on neuroglobin and cytochrome c interactions.

Phycocyanin is an excellent antioxidant with anti-inflammatory effects on which recent studies are growing; however, its specific target remains unclear. Linear tetrapyrrole compounds such as bilirubin have been shown to lead to the induction of heme oxygenase 1 expression in vivo, thus achieving antioxidant and anti-inflammatory effects. Phycocyanin is bound internally with linear tetrapyrrole phycocyanobilin in a similar structure to bilirubin. We speculate that there is probably a way of inducing the expression of heme oxygenase 1, with which tissue oxidative stress and inflammation can be inhibited, thus inhibiting pulmonary fibrosis caused by oxidative damage and inflammation of lung. By optimizing the enzymatic hydrolysis process, phycocyanobilin-bound phycocyanin peptide were obtained, and its in vitro antioxidant, anti-inflammatory, and anti-pulmonary fibrosis activities were investigated. The results show that the phycocyanobilin peptide was able to alleviate oxidative and inflammatory damage in cells through the Keap1-Nrf2-HO-1 pathway, which in turn relieved pulmonary fibrosis symptoms.

The intracellular concentrations of oxygen and reactive oxygen species (ROS) in living cells represent critical information for investigating physiological and pathological conditions. Real-time measurement often relies on genetically encoded proteins that are responsive to fluctuations in either oxygen or ROS concentrations. The direct binding or chemical reactions that occur in their presence either directly alter the fluorescence properties of the binding protein or alter the fluorescence properties of fusion partners, mostly consisting of variants of the green fluorescent protein. Oxygen sensing takes advantage of several mechanisms, including (i) the oxygen-dependent hydroxylation of a domain of the hypoxia-inducible factor-1, which, in turn, promotes its cellular degradation along with fluorescent fusion partners; (ii) the naturally oxygen-dependent maturation of the fluorophore of green fluorescent protein variants; and (iii) direct oxygen binding by proteins, including heme proteins, expressed in fusion with fluorescent partners, resulting in changes in fluorescence due to conformational alterations or fluorescence resonance energy transfer. ROS encompass a group of highly reactive chemicals that can interconvert through various chemical reactions within biological systems, posing challenges for their selective detection through genetically encoded sensors. However, their general reactivity, and particularly that of the relatively stable oxygen peroxide, can be exploited for ROS sensing through different mechanisms, including (i) the ROS-induced formation of disulfide bonds in engineered fluorescent proteins or fusion partners of fluorescent proteins, ultimately leading to fluorescence changes; and (ii) conformational changes of naturally occurring ROS-sensing domains, affecting the fluorescence properties of fusion partners. In this review, we will offer an overview of these genetically encoded biosensors.

Heme is a ubiquitous and essential iron containing metallo-organic cofactor required for virtually all aerobic life. Heme synthesis is initiated and completed in mitochondria, followed by certain covalent modifications and/or its delivery to apo-hemoproteins residing throughout the cell. While the biochemical aspects of heme biosynthetic reactions are well understood, the trafficking of newly synthesized heme—a highly reactive and inherently toxic compound—and its subsequent delivery to target proteins remain far from clear. In this review, we summarize current knowledge about heme biosynthesis and trafficking within and outside of the mitochondria.

Introduction Neutrophil extracellular traps (NETs) are DNA scaffolds enriched with antimicrobial proteins. NETs have been implicated in the development of various diseases, such as cardiovascular disease. Here, we investigate the association of demographic and cardiovascular (CVD) risk factors with NETs in the general population. Material and methods Citrated plasma was collected from 6449 participants, aged [greater than or equal to]55 years, as part of the prospective population-based Rotterdam Study. NETs were quantified by measuring MPO-DNA complex using an ELISA. We used linear regression to determine the associations between MPO-DNA complex and age, sex, cardio-metabolic risk factors, and plasma markers of inflammation and coagulation. Results MPO-DNA complex levels were weakly associated with age (log difference per 10 year increase: -0.04 mAU/mL, 95% confidence interval [CI] -0.06;-0.02), a history of coronary heart disease (yes versus no: -0.10 mAU/mL, 95% CI -0.17;-0.03), the use of lipid-lowering drugs (yes versus no: -0.06 mAU/mL, 95% CI -0.12;-0.01), and HDL-cholesterol (per mmol/l increase: -0.07 mAU/mL/, 95% CI -0.12;-0.03). Conclusions Older age, a history of coronary heart disease, the use of lipid-lowering drugs and higher HDL-cholesterol are weakly correlated with lower plasma levels of NETs. These findings show that the effect of CVD risk factors on NETs levels in a general population is only small and may not be of clinical relevance. This supports that NETs may play a more important role in an acute phase of disease than in a steady state situation.

Neutrophil extracellular traps (NETs) are DNA scaffolds enriched with antimicrobial proteins. NETs have been implicated in the development of various diseases, such as cardiovascular disease. Here, we investigate the association of demographic and cardiovascular (CVD) risk factors with NETs in the general population. Citrated plasma was collected from 6449 participants, aged [greater than or equal to]55 years, as part of the prospective population-based Rotterdam Study. NETs were quantified by measuring MPO-DNA complex using an ELISA. We used linear regression to determine the associations between MPO-DNA complex and age, sex, cardio-metabolic risk factors, and plasma markers of inflammation and coagulation. MPO-DNA complex levels were weakly associated with age (log difference per 10 year increase: -0.04 mAU/mL, 95% confidence interval [CI] -0.06;-0.02), a history of coronary heart disease (yes versus no: -0.10 mAU/mL, 95% CI -0.17;-0.03), the use of lipid-lowering drugs (yes versus no: -0.06 mAU/mL, 95% CI -0.12;-0.01), and HDL-cholesterol (per mmol/l increase: -0.07 mAU/mL/, 95% CI -0.12;-0.03). Older age, a history of coronary heart disease, the use of lipid-lowering drugs and higher HDL-cholesterol are weakly correlated with lower plasma levels of NETs. These findings show that the effect of CVD risk factors on NETs levels in a general population is only small and may not be of clinical relevance. This supports that NETs may play a more important role in an acute phase of disease than in a steady state situation.

Nitric oxide (NO) is an active and critical nitrogen oxide in the microbe-driven nitrogen biogeochemical cycle, and is of great interest to medicine and the biological sciences. As a gas molecule prior to oxygen, NO respiration represents an early form of energy generation via various reactions in prokaryotes. Major enzymes for endogenous NO formation known to date include two types of nitrite reductases in denitrification, hydroxylamine oxidoreductase in ammonia oxidation, and NO synthases (NOSs). While the former two play critical roles in shaping electron transport pathways in bacteria, NOSs are intracellular enzymes catalyzing metabolism of certain amino acids and have been extensively studied in mammals. NO interacts with numerous cellular targets, most of which are redox-active proteins. Doing so, NO plays harmful and beneficial roles by affecting diverse biological processes within bacterial physiology. Here, we discuss recent advances in the field, including NO-forming enzymes, the molecular mechanisms by which these enzymes function, physiological roles of bacterial NOSs, and regulation of NO homeostasis in bacteria.

Ligand–protein docking aims to predict how a ligand binds to a biological macromolecule and is a fundamental technique in structure-based computer-aided drug design. However, accurately modeling covalent binding, metal coordination, and polarization effects remains challenging for classical docking algorithms. Here, we present an extension of our Attracting Cavities docking algorithm that enables hybrid quantum mechanical/molecular mechanical (QM/MM) calculations at various levels of theory. To evaluate its performance, we benchmarked the method on three diverse datasets covering non-covalent drug–target complexes, covalent complexes, and hemoprotein complexes, using both semi-empirical and density functional theory approaches. The results demonstrate that QM/MM docking is especially advantageous for metal-binding complexes, where the fast semi-empirical PM7 method yields a significant improvement over classical docking. When describing the active site residues at the density functional theory level, dispersion corrections are crucial for meaningful energies. Overall, the QM/MM method outperforms the classical approach for metalloproteins, performs comparably for covalent complexes, and shows slightly lower success rates for non-covalent complexes.

The structure–function relationship is at the heart of biology, and major protein deformations are correlated to specific functions. For ferrous heme proteins, doming is associated with the respiratory function in hemoglobin and myoglobins. Cytochrome c (Cyt c) has evolved to become an important electron-transfer protein in humans. In its ferrous form, it undergoes ligand release and doming upon photoexcitation, but its ferric form does not release the distal ligand, while the return to the ground state has been attributed to thermal relaxation. Here, by combining femtosecond Fe Kα and Kβ X-ray emission spectroscopy (XES) with Fe K-edge X-ray absorption near-edge structure (XANES), we demonstrate that the photocycle of ferric Cyt c is entirely due to a cascade among excited spin states of the iron ion, causing the ferric heme to undergo doming, which we identify. We also argue that this pattern is common to a wide diversity of ferric heme proteins, raising the question of the biological relevance of doming in such proteins.