Mercury(II) complexes which have a bulky cholyl amide group at the ortho or para position of benzenethiolate, Hg[S-2-{C23H36(OH)3}CONHC6H4]2 (1) with an intramolecular NH···S hydrogen bond and Hg[S-4-{C23H36(OH)3}CONHC6H4]2 (2), were synthesized to prepare an aqueous micellar solution. A hydrated Hg(II) ion was formed from the Hg(II) thiolate complexes, 1 and 2, at the ligand dissociation point (pH 4.0 and 4.9, respectively) near the pKa values (5.7 and 7.0, respectively) of the corresponding thiols. The hydrated Hg(II) ion was detected by the formation of Hg(0) species reduced with Na2S2O4 in an aqueous micellar solution. The NH···S hydrogen bond lowers the pKa value of the conjugated thiol to protect the Hg−S bond from dissociation by water under neutral conditions.
Aberrant immune responses to viral pathogens contribute to pathogenesis, but our understanding of pathological immune responses caused by viruses within the human virome, especially at a population scale, remains limited. We analyzed whole-genome sequencing datasets of 6,321 Japanese individuals, including patients with autoimmune diseases (psoriasis vulgaris, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), pulmonary alveolar proteinosis (PAP) or multiple sclerosis) and coronavirus disease 2019 (COVID-19), or healthy controls. We systematically quantified two constituents of the blood DNA virome, endogenous HHV-6 (eHHV-6) and anellovirus. Participants with eHHV-6B had higher risks of SLE and PAP; the former was validated in All of Us. eHHV-6B-positivity and high SLE disease activity index scores had strong correlations. Genome-wide association study and long-read sequencing mapped the integration of the HHV-6B genome to a locus on chromosome 22q. Epitope mapping and single-cell RNA sequencing revealed distinctive immune induction by eHHV-6B in patients with SLE. In addition, high anellovirus load correlated strongly with SLE, RA and COVID-19 status. Our analyses unveil relationships between the human virome and autoimmune and infectious diseases. Analysis of the blood DNA virome in patients with COVID-19 and autoimmune disease associates endogenous HHV-6 (eHHV-6) and high anellovirus load with increased disease risk, most notably for systemic lupus erythematosus. eHHV-6 carriers show a distinct immune response.
Artificial metalloenzyme, composed of metal complex(es) and a host protein, is a promising way to mimic enzyme catalytic functions or develop novel enzyme-like catalysis. However, it is highly challenging to unveil the active site and exact reaction mechanism inside artificial metalloenzyme, which is the bottleneck in its rational design. We present a QM/MM study of the complicated reaction mechanism for the recently developed artificial metalloenzyme system, (Rh(nbd)·apo-Fr) (nbd = norbornadiene), which is composed of a rhodium complex [Rh(nbd)Cl](2) and the recombinant horse L-chain apo-Ferritin. We found that binding sites suggested by the X-ray crystal structure, i.e., sites A, B, and C, are only precursors/intermediates, not true active sites for polymerization of phenylacetylene (PA). A new hydrophobic site, which we name D, is suggested to be the most plausible active site for polymerization. Active site D is generated after coordination of first monomer PA by extrusion of the Rh(I)(PA) complex to a hydrophobic pocket near site B. Polymerization occurs in site D via a Rh(I)-insertion mechanism. A specific "hydrophobic region" composed by the hydrophobic active site D, the nonpolar 4-fold channel, and other hydrophobic residues nearby is found to facilitate accumulation, coordination, and insertion of PA for polymerization. Our results also demonstrate that the hydrophobic active site D can retain the native regio- and stereoselectivity of the Rh-catalyzed polymerization of PA without protein. This study highlights the importance of theoretical study in mechanistic elucidation and rational design of artificial metalloenzymes, indicating that even with X-ray crystal structures at hand we may still be far from fully understanding the active site and catalytic mechanism of artificial metalloenzymes.
The F43W/H64L myoglobin mutant was previously constructed to investigate the effects of electron-rich tryptophan residue in the heme vicinity on the catalysis, where we found that Trp-43 in the mutant was oxidatively modified in the reaction with m-chloroperbenzoic acid (mCPBA). To identify the exact structure of the modified tryptophan in this study, the mCPBA-treated F43W/H64L mutant has been digested stepwise with Lys-C achromobacter and trypsin to isolate two oxidation products by preparative fast protein liquid chromatography. The close examinations of the 1H NMR spectra of peptide fragments reveal that two forms of the modified tryptophan must have 2,6-disubstituted indole substructures. The 13C NMR analysis suggests that one of the modified tryptophan bears a unique hydroxyl group in stead of the NH2 group at the amino-terminal. The results together with mass spectrometry (MS)/MS analysis (30 Da increase in mass of Trp-43) indicate that oxidation products of Trp-43 are 2,6-dihydro-2,6-dioxoindole and 2,6-dihydro-2-imino-6-oxoindole derivatives. Our finding is the first example of the oxidation of aromatic carbons by the myoglobin mutant system.
The Cover artwork summarizes the principal themes represented by the current issue on Artificial Metalloenzymes. For an introduction to this issue and its main areas of focus, please see the Guest Editorial by T. Ueno on page 13.
Novel organometallic proteins have been synthesized as a 1:1 composite of rhodium 2,6-bis(2-oxazolinyl)phenyl (Rh·Phebox) complexes with the apo-form of myoglobin, which is an oxygen-transport protein having b-type heme (iron porphyrin IX) as a natural prosthetic group. The X-ray structural analysis reveals that the Rh·Phebox complex with phenyl substituents is included in the cavity with an almost perpendicular arrangement to that of the heme. The unique arrangement is supported by hydrogen bonding and a number of hydrophobic interactions, especially π−π interactions between the phenyl rings of the substituents and the imidazole moiety of His93, which is a ligand of the rhodium atom. Semiquantitative analysis of the composite stability by ESI mass spectroscopy clearly indicates that the stability of the composites depends on an extent of the interaction between the substituents of the Rh·Phebox complexes and His93. Enantioselectivity for the chiral (S,S)- and (R,R)-Rh·Phebox complexes with the phenyl substituents is also observed in terms of the difference in the stability of the composites. According to the arrangement of the amino acid residues around the Phebox ligand, the (S,S)-form has a suitable configuration to fit one of its phenyl rings into the hollow formed by Leu89, His93, and Ile99, while structural distortion will result if the (R,R)-isomer adopts an arrangement similar to that of the (S,S)-isomer, which is confirmed with the enhancement of the maximal and minimal ellipticities of the circular dichroic spectrum in comparison with that of the intact (R,R)-isomer. Such structural strain would reduce the efficient π−π interaction between the ligand substituents and His93, resulting in less stability of the composite containing the (R,R)-isomer. The results obtained here demonstrate that the myoglobin cavity is capable of accommodating organometallic compounds totally different from the heme in its molecular shape and the arrangement in the cavity. The extension and application of the present method will allow us to produce artificial organometallic proteins bearing functions that are difficult to achieve with natural prosthetic groups.
