Oxygen activation occurs at a wide variety of enzyme active sites. Mechanisms previously proposed for the copper monooxygenase, dopamine -monooxygenase (DM), involve the accumulation of an activated oxygen intermediate with the properties of a copper-peroxo or copper-oxo species before substrate activation. These are reminiscent of the mechanism of cytochrome P-450, where a heme iron stabilizes the activated O 2 species. Herein, we report two experimental probes of the activated oxygen species in DM. First, we have synthesized the substrate analog, ,-difluorophenethylamine, and examined its capacity to induce reoxidation of the prereduced copper sites of DM upon mixing with O 2 under rapid freeze-quench conditions. This experiment fails to give rise to an EPR-detectable copper species, in contrast to a substrate with a C-H active bond. This indicates either that the reoxidation of the enzyme-bound copper sites in the presence of O 2 is tightly linked to C-H activation or that a diamagnetic species Cu(II)-O 2 ⅐ ⅐ has been formed. In the context of the open and fully solvent-accessible active site for the homologous peptidylglycine-␣-hydroxylating monooxygenase and by analogy to cytochrome P-450, the accumulation of a reduced and activated oxygen species in DM before C-H cleavage would be expected to give some uncoupling of oxygen and substrate consumption. We have, therefore, examined the degree to which O 2 and substrate consumption are coupled in DM using both end point and initial rate experimental protocols. With substrates that differ by more than three orders of magnitude in rate, we fail to detect any uncoupling of O 2 uptake from product formation. We conclude that there is no accumulation of an activated form of O 2 before C-H abstraction in the DM and peptidylglycine-␣-hydroxylating monooxygenase class of copper monooxygenases, presenting a mechanism in which a diamagnetic Cu(II)-superoxo complex, formed initially at very low levels, abstracts a hydrogen atom from substrate to generate Cu(II)-hydroperoxo and substrate-free radical as intermediates. Subsequent participation of the second copper site per subunit completes the reaction cycle, generating hydroxylated product and water. Dopamine -monooxygenase (DM) 1 along with peptidylglycine-␣-hydroxylating monooxygenase (PHM) comprise a unique class of enzymes that contain only copper as a cofactor and catalyze the cleavage of O 2 to form hydroxylated product and water. DM is of central importance in the catecholamine biosynthetic pathway, catalyzing the conversion of dopamine to norepinephrine (Scheme 1, top), where both substrate and product serve as neurotransmitters within the central nervous system (1). Primarily localized within the secretory granules of adrenal chromaffin cells and neurons, DM is a large, tetrameric glycoprotein (75 kDa per monomer) consisting of two disulfide-linked dimers.Although no crystal structure has been reported, extensive structural data exist for DM. Extended X-ray absorption fine structure was used to char...
The waning efficacy of SARS-CoV-2 vaccines, combined with the continued emergence of variants resistant to vaccine-induced immunity, has reignited debate over the need for booster vaccine doses. To address this, we examined the neutralizing antibody response against the spike protein of five major SARS-CoV-2 variants, D614G, Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529), in health care workers (HCWs) vaccinated with SARS-CoV-2 mRNA vaccines. Serum samples were collected pre-vaccination, three weeks post-first vaccination, one month post-second vaccination, and six months post-second vaccination. Minimal neutralizing antibody titers were detected against Omicron pseudovirus at all four time points, including for a majority of patients who had SARS-CoV-2 breakthrough infections. Neutralizing antibody titers against all other variant spike protein-bearing pseudoviruses declined dramatically from one to six months after the second mRNA vaccine dose, although SARS-CoV-2 infection boosted vaccine responses. Additionally, mRNA-1273-vaccinated HCWs exhibited about two-fold higher neutralizing antibody titers than BNT162b2-vaccinated HCWs. Together these results demonstrate possible waning of antibody-mediated protection against SARS-CoV-2 variants that is dependent on prior infection status and the mRNA vaccine received. They also show that the Omicron variant spike protein can almost completely escape from neutralizing antibodies elicited in recipients of only two mRNA vaccine doses.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell–cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell–cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.
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