Brian Bennett

Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples

Imaging volumes as thick as whole cells at three-dimensional (3D) super-resolution is required to reveal unknown features of cellular organization. We report a light microscope that generates images with translationally invariant 30 × 30 × 75nm resolution over a depth of several micrometers. This method, named biplane (BP) FPALM, combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) enabling 3D sub-diffraction resolution without compromising speed or sensitivity.

Sulfite : Cytochrome c oxidoreductase from Thiobacillus novellus - Purification, characterization, and molecular biology of a heterodimeric member of the sulfite oxidase family

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC1.8.2.1). Here we show that the enzyme from Thiobacillus novellus is a periplasmically located αβ heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa mono-heme cytochrome c 552 subunit (midpoint redox potential, E m8.0 = +280 mV). The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K m values for sulfite and cytochrome c 550 were determined to be 27 and 4 μm, respectively; the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorABgenes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream ofsorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any knownc-type cytochromes.

H2AX chromatin structures and their response to DNA damage revealed by 4Pi microscopy

DNA double-strand breaks (DSBs) caused by cellular exposure to genotoxic agents or produced by inherent metabolic processes initiate a rapid and highly coordinated series of molecular events resulting in DNA damage signaling and repair. Phosphorylation of histone H2AX to form γ-H2AX is one of the earliest of these events and is important for coordination of signaling and repair activities. An intriguing aspect of H2AX phosphorylation is that γ-H2AX spreads a limited distance up to 1–2 Mbp from the site of a DNA break in mammalian cells. However, neither the distribution of H2AX throughout the genome nor the mechanism that defines the boundary of γ-H2AX spreading have yet been described. Here, we report the identification of previously undescribed H2AX chromatin structures by successfully applying 4Pi microscopy to visualize endogenous nuclear proteins. Our observations suggest that H2AX is not distributed randomly throughout bulk chromatin, rather it exists in distinct clusters that themselves are uniformly distributed within the nuclear volume. These data support a model in which the size and distribution of H2AX clusters define the boundaries of γ-H2AX spreading and also may provide a platform for the immediate and robust response observed after DNA damage.

Role of the Zn1 and Zn2 sites in Metallo-β-lactamase L1

In an effort to probe the role of the Zn(II) sites in metallo-beta-lactamase L1, mononuclear metal ion containing and heterobimetallic analogues of the enzyme were generated and characterized using kinetic and spectroscopic studies. Mononuclear Zn(II)-containing L1, which binds Zn(II) in the consensus Zn1 site, was shown to be slightly active; however, this enzyme did not stabilize a nitrocefin-derived reaction intermediate that had been previously detected. Mononuclear Co(II)- and Fe(III)-containing L1 were essentially inactive, and NMR and EPR studies suggest that these metal ions bind to the consensus Zn2 site in L1. Heterobimetallic analogues (ZnCo and ZnFe) analogues of L1 were generated, and stopped-flow kinetic studies revealed that these enzymes rapidly hydrolyze nitrocefin and that there are large amounts of the reaction intermediate formed during the reaction. The heterobimetallic analogues were reacted with nitrocefin, and the reactions were rapidly freeze quenched. EPR studies on these samples demonstrate that Co(II) is 5-coordinate in the resting state, proceeds through a 4-coordinate species during the reaction, and is 5-coordinate in the enzyme-product complex. These studies demonstrate that the metal ion in the Zn1 site is essential for catalysis in L1 and that the metal ion in the Zn2 site is crucial for stabilization of the nitrocefin-derived reaction intermediate.

Trapping and Characterization of a Reaction Intermediate in Carbapenem Hydrolysis by B. cereus Metallo-β-lactamase

Metallo-beta-lactamases hydrolyze most beta-lactam antibiotics. The lack of a successful inhibitor for them is related to the previous failure to characterize a reaction intermediate with a clinically useful substrate. Stopped-flow experiments together with rapid freeze-quench EPR and Raman spectroscopies were used to characterize the reaction of Co(II)-BcII with imipenem. These studies show that Co(II)-BcII is able to hydrolyze imipenem in both the mono- and dinuclear forms. In contrast to the situation met for penicillin, the species that accumulates during turnover is an enzyme-intermediate adduct in which the beta-lactam bond has already been cleaved. This intermediate is a metal-bound anionic species with a novel resonant structure that is stabilized by the metal ion at the DCH or Zn2 site. This species has been characterized based on its spectroscopic features. This represents a novel, previously unforeseen intermediate that is related to the chemical nature of carbapenems, as confirmed by the finding of a similar intermediate for meropenem. Since carbapenems are the only substrates cleaved by B1, B2, and B3 lactamases, identification of this intermediate could be exploited as a first step toward the design of transition-state-based inhibitors for all three classes of metallo-beta-lactamases.

Structural studies on a mitochondrial glyoxalase II.

Glyoxalase 2 is a β-lactamase fold-containing enzyme that appears to be involved with cellular chemical detoxification. Although the cytoplasmic isozyme has been characterized from several organisms, essentially nothing is known about the mitochondrial proteins. As a first step in understanding the structure and function of mitochondrial glyoxalase 2 enzymes, a mitochondrial isozyme (GLX2-5) from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and 1H NMR spectroscopies, and x-ray crystallography. The recombinant enzyme was shown to bind 1.04 ± 0.15 eq of iron and 1.31 ± 0.05 eq of Zn(II) and to exhibit kcat and Km values of 129 ± 10 s-1 and 391 ± 48 μm, respectively, when using S-d-lactoylglutathione as the substrate. EPR spectra revealed that recombinant GLX2-5 contains multiple metal centers, including a predominant Fe(III)Z-n(II) center and an anti-ferromagnetically coupled Fe(III)Fe(II) center. Unlike cytosolic glyoxalase 2 from A. thaliana, GLX2-5 does not appear to specifically bind manganese. 1H NMR spectra revealed the presence of at least eight paramagnetically shifted resonances that arise from protons in close proximity to a Fe(III)Fe(II) center. Five of these resonances arose from solvent-exchangeable protons, and four of these have been assigned to NH protons on metal-bound histidines. A 1.74-Å resolution crystal structure of the enzyme revealed that although GLX2-5 shares a number of structural features with human GLX2, several important differences exist. These data demonstrate that mitochondrial glyoxalase 2 can accommodate a number of different metal centers and that the predominant metal center is Fe(III)Zn(II).

The Metallo-β-lactamase GOB is a mono-Zn(II) enzyme with a novel active site

Metallo-β-lactamases (MβLs) are zinc-dependent enzymes able to hydrolyze and inactivate most β-lactam antibiotics. The large diversity of active site structures and metal content among MβLs from different sources has limited the design of a pan-MβL inhibitor. Here we report the biochemical and biophysical characterization of a novel MβL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MβLs. Contrasting all other related MβLs, GOB-18 is fully active against a broad range of β-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MβLs.

Evidence for a Dinuclear Active Site in the Metallo-β-lactamase BcII with Substoichiometric Co(II) A NEW MODEL FOR METAL UPTAKE

Metallo-β-lactamases are zinc-dependent enzymes that constitute one of the main resistance mechanisms to β-lactam antibiotics. Metallo-β-lactamases have been characterized both in mono- and dimetallic forms. Despite many studies, the role of each metal binding site in substrate binding and catalysis is still unclear. This is mostly due to the difficulties in assessing the metal content and site occupancy in solution. For this reason, Co(II) has been utilized as a useful probe of the active site structure. We have employed UV-visible, EPR, and NMR spectroscopy to study Co(II) binding to the metallo-β-lactamase BcII from Bacillus cereus. The spectroscopic features were attributed to the two canonical metal binding sites, the 3H (His116, His118, and His196) and DCH (Asp120, Cys221, and His263) sites. These data clearly reveal the coexistence of mononuclear and dinuclear Co(II)-loaded forms at Co(II)/enzyme ratios as low as 0.6. This picture is consistent with the macroscopic dissociation constants here determined from competition binding experiments. A spectral feature previously assigned to the DCH site in the dinuclear species corresponds to a third, weakly bound Co(II) site. The present work emphasizes the importance of using different spectroscopic techniques to follow the metal content and localization during metallo-β-lactamase turnover.

Differential Binding of Co(II) and Zn(II) to Metallo-β-Lactamase Bla2 from Bacillus anthracis

In an effort to probe the structure, mechanism, and biochemical properties of metallo-beta-lactamase Bla2 from Bacillus anthracis, the enzyme was overexpressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equiv of Zn(II). Steady-state kinetic studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, di-Co(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analogue. Rapid kinetics and UV-vis, (1)H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distributed, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms, underscoring the need to demonstrate transferability when extrapolating results on Co(II)-substituted proteins to the native Zn(II)-containing forms.

Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM‑1

In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV–vis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data.