Journal of Biological Inorganic Chemistry最新文献

Protein film electrochemistry has helped to unravel many complex reactivities of electron-transferring proteins and enzymes. A versatile descendant, the 'Electrochemical Leaf', offers new opportunities to extend electrochemical control to myriad enzymes that neither transfer electrons nor catalyse any redox reaction, including those dependent on spectroscopically limited, labile or other challenging metal ions. By embedding a cascade comprised of several enzymes-one of which electrochemically recycles NAD(P)(H), a second being a dehydrogenase-within a porous electrode formed from fused nanoparticles, the interconnected reactions are tightly channeled to transmit energy and information, rapidly and interactively. Under nanoconfinement, nicotinamide cofactors and cascade intermediates serve as specific current carriers, far beyond the electron itself.

Two related classes of ligand-binding heme c-containing proteins with a high degree of structural homology have been identified and characterized over recent decades: cytochromes P460 (cyts P460), defined by an unusual heme-lysine cross-link, and cytochromes c'-β (cyts c'-β), containing a canonical c-heme without the lysine cross-link. The shared protein fold of the cyt P460-cyt c'-β superfamily can accommodate a variety of heme environments with entirely different reactivities. On the one hand, cyts P460 with polar distal pockets have been shown to oxidize NH₂OH to NO and/or N₂O via proton-coupled electron transfer. On the other hand, cyts c'-β with hydrophobic distal pockets have a proposed gas binding function similar to the unrelated, but more extensively characterized, alpha helical cytochromes c'. Recent studies have also identified 'halfway house' proteins (cyts P460 with non-polar heme pockets and cyts c'-β with polar distal heme pockets) with functions yet to be resolved. Here, we review the structural, spectroscopic and enzymatic properties of the cyt P460-cyt c'-β superfamily with a view to understanding the structural determinants of their different functional properties.

Bacteria and fungi produce natural products that coordinate copper for a variety of functions. Many copper-binding natural products function as copper-chelating metallophores, or chalkophores, that scavenge copper from the environment to meet cellular needs. By contrast, some compounds sequester toxic levels of environmental copper to protect the producing microorganism. These copper-binding compounds often have antimicrobial activities as well. In recent years, a number of new copper-coordinating natural products have been reported, including both ribosomally and non-ribosomally synthesized molecules. There have also been significant advances in understanding the biosynthesis of these and previously known copper chelators, leading to the discovery of new enzyme families. This review summarizes the recently discovered copper-binding natural products, their biosynthetic pathways, and their functions. By highlighting key biosynthetic enzymes, we hope to inspire the discovery of new copper-coordinating natural products that may be used as therapeutics and antimicrobial agents.

PnpC1C2 is an enzyme from the soil bacterium Pseudomonas putida DLL-E4 that is in the pathway for the oxidative catabolism of 4-nitrophenol. PnpC1C2 oxidatively cleaves hydroquinone into γ-hydroxymuconic semialdehyde. It belongs to the type II hydroquinone dioxygenase family, a relatively uncharacterized group of mononuclear non-heme Fe(II)-dependent enzymes that catalyze oxidative ring-cleavage reactions, which includes the well-studied catechol extradiol dioxygenases as well as the structurally unrelated 2,6-dichlorohydroquinone dioxygenase (PcpA). Steady-state kinetics studies using UV/Vis spectroscopy were performed to characterize the enzyme specificity towards various substituted hydroquinones. In addition to its native substrate, PnpC1C2 was active towards a variety of monosubstituted hydroquinones. Methyl- and methoxyhydroquinone showed a moderately higher $K_{\mathrm{mA}}^{\mathrm{app}}$ , and chloro- and bromohydroquinone showed a moderately lower $k_{\mathrm{cat}}^{\mathrm{app}}$ , but all had a $k_{\mathrm{cat}}^{\mathrm{app}}\left(\phantom{k_{\mathrm{cat}}^{\mathrm{app}}{K}_{\mathrm{mA}}^{\mathrm{app}}}\right)K_{\mathrm{mA}}^{\mathrm{app}}$ within an order of magnitude of unsubstituted hydroquinone. Likewise, only small differences in the rates of mechanism-based inactivation were observed among these substrates. Among disubstituted hydroquinones, only 2,6- and 2,5-dimethylhydroquinone showed any activity, with the latter only barely detectable. A variety of para-substituted phenols were found to be good inhibitors of PnpC1C2. NMR studies were performed to determine the regioselectivity of ring-cleavage with monosubstituted hydroquinones. All monosubstituted hydroquinones tested (methyl-, chloro-, bromo-, and methoxyhydroquinone) yielded exclusively the 1,6-cleavage product. Thus, PnpC1C2 shows notable differences in both its substrate specificity and the ring-cleavage regioselectivity compared to that of PcpA. These results provide an important basis for future comparison of structure-function correlations among the hydroquinone ring-cleaving dioxygenases.

In the reaction of aquacobalamin (aquaCbl) with chlorite, a stable species is detected and assigned as a Co(III)-chlorite complex, Co(III)-OClO^-. Its UV-Vis spectrum is almost identical to that of aquaCbl, except for some minor differences at ~ 430 nm; cyanide can eliminate and prevent these changes. The ¹H-NMR spectra reveal strong influences of chlorite on the B2 and B4 protons of the cobalt-bound dimethyl benzimidazole ligand. Together, the UV-Vis and NMR titrations suggest a Kd of 10 mM or higher for chlorite on Cbl. Resonance Raman spectra reveal minor changes in the spectrum of aquaCbl to chlorite-as well as a disappearance of the free chlorite signals, consistent with Cbl-chlorite complex formation. Corroboration for these interpretations is also offered from mass spectrometry and DFT calculations. This Co(III)-OClO^- complex would be a stable analogue of the first reaction intermediate in the catalytic cycle of chlorite dismutase, or in the reaction of chlorite with a number of other heme proteins. The differences in reactivity between Co(III) cobalamin and Fe(III) heme towards chlorite are analyzed and rationalized, leading to a reconciliation of experimental and computational data for the latter.

The aim of this study was to elucidate the potential mechanism of the antihyperglycemic action of the donut-shaped Preyssler-Pope-Jeannin polyanion salt (NH₄)₁₄[NaP₅W₃₀O₁₁₀] 31H₂O (NaP₅W₃₀) and its effect on metabolic disorders associated with diabetes. For this purpose, relevant parameters of blood glucose regulation, lipid profile, and electrolyte status were monitored in streptozotocin (STZ)-induced diabetic rats that were orally treated with 20 mg/kg/day NaP₅W₃₀ for three weeks. The serum insulin concentration was increased in diabetic animals treated with NaP₅W₃₀ (20 mg/kg/day, per os, three weeks), which could be one of the possible mechanisms of the confirmed antihyperglycemic effect. In addition, the administration of NaP₅W₃₀ significantly reduced hyperglycemia and glycated haemoglobin A_1c (HbA_1c) in STZ-induced diabetic rats, although normoglycemic values were not achieved. Furthermore, a statistically significant 1.3-fold reduction in serum total cholesterol and a 1.7-fold reduction in high-density lipoprotein (HDL) cholesterol were observed in the NaP₅W₃₀ treatment group compared to the diabetic control group. In contrast, NaP₅W₃₀ had no effect on homeostasis model assessment of insulin resistance (HOMA-IR) index values, electrolyte concentrations, or serum concentrations of low-density lipoprotein (LDL) cholesterol, apolipoprotein A1 (Apo A1), apolipoprotein B (Apo B), or total triglycerides. In summary, NaP₅W₃₀ effectively improved glycoregulation in diabetic rats via the considerable stimulation of insulin as a putative mechanism. Moreover, NaP₅W₃₀ did not affect rat weight or disrupt lipid and electrolyte status, common diabetes-followed side effects and risk factors for various life-threatening complications. Thus, NaP₅W₃₀ could be considered a promising antidiabetic drug-candidate that deserves further investigation.

NiaR is a regulatory protein that represses the expression of proteins involved in the de novo biosynthesis and uptake of nicotinic acid (NA), with NA acting as a co-repressor. The previously published structure of NiaR from Thermotoga maritima (TmNiaR) identified it as a functional homodimer containing a transition metal ion in a suspected NA-binding pocket. Here, we present the crystal structure of NA bound to the iron-metalated form of TmNiaR. Supported by spectroscopic and solution studies, this structure shows that NA binds to a protein-bound ferrous ion via its ring nitrogen. In addition, the carboxylate group on NA interacts with Tyr108 from the dyad-related subunit, repositioning the likely DNA-binding domains of the dimer to promote high-affinity interactions with DNA operators. The specificity of TmNiaR for NA can be explained by the hydrogen bonding scheme within the NA-binding pocket.

Native metalloenzymes are unparalleled in their ability to perform efficient small molecule activation reactions, converting simple substrates into complex products. Most of these natural systems possess multiple metallocofactors to facilitate electron transfer or cascade catalysis. While the field of artificial metalloenzymes is growing at a rapid rate, examples of artificial enzymes that leverage two distinct cofactors remain scarce. In this work, we describe a new class of artificial enzymes containing two different metallocofactors, incorporated through bioorthogonal strategies. Nickel-substituted rubredoxin (Ni^Rd), which is a structural and functional mimic of [NiFe] hydrogenases, is used as a scaffold. Incorporation of a synthetic bimetallic inorganic complex based on a macrocyclic biquinazoline ligand (M^MBQ) was accomplished using a novel chelating thioether linker. Neither the structure of the Ni^Rd active site nor the M^MBQ were altered upon attachment, and each site retained independent redox activity. Electrocatalysis was observed from each site, with the switchability of the system demonstrated through the use of catalytically inert metal centers. This M^MBQ-Ni^Rd platform offers a new avenue to create multicofactor artificial metalloenzymes in a robust system that can be easily tuned both through modifications to the protein scaffold and the synthetic moiety, with applications for redox catalysis and tandem reactivity.

Iron-sulfur proteins perform a wide variety of reactions central to the metabolisms of all living organisms. Foundational to their reaction chemistry are the rich electronic structures of their constituent Fe-S clusters, which differ in important ways from the active sites of mononuclear Fe enzymes. In this perspective, we summarize the essential electronic structure features that make Fe-S clusters unique, and point to the need for studies aimed at understanding the electronic basis for their reactivity under physiological conditions. Specifically, at ambient temperature, both the ground state and a large number of excited states are thermally populated, and thus a complete understanding of Fe-S cluster reactivity must take into account the properties, energies, and reactivity patterns of these excited states. We highlight prior research toward characterizing the low-energy excited states of Fe-S clusters that has established what is now a consensus model of these excited state manifolds and the bonding interactions that give rise to them. In particular, we discuss the low-energy alternate spin states and valence electron configurations that occur in Fe-S clusters of varying nuclearities, and finally suggest that there may be unrecognized functional roles for these states.