Striking Improvement in Peroxidase Activity of Cytochrome c by Modulating Hydrophobicity of Surface‐Functionalized Gold Nanoparticles within Cationic Reverse Micelles

This work demonstrates a remarkable enhancement in the peroxidase activity of mitochondrial membrane protein cytochrome c (cyt c) by perturbing its tertiary structure in the presence of surface‐functionalised gold nanoparticles (GNPs) within cetyltrimethylammonium bromide (CTAB) reverse micelles. The loss in the tertiary structure of cyt c exposes its heme moiety (which is buried inside in the native globular form), which provides greater substrate (pyrogallol and H₂O₂) accessibility to the reactive heme residue. The surfactant shell of the CTAB reverse micelle in the presence of co‐surfactant (n‐hexanol) exerted higher crowding effects on the interfacially bound cyt c than similar anionic systems. The congested interface led to protein unfolding, which resulted in a 56‐fold higher peroxidase activity of cyt c than that in water. Further perturbation in the protein’s structure was achieved by doping amphiphile‐capped GNPs with varying hydrophobicities in the water pool of the reverse micelles. The hydrophobic moiety on the surface of the GNPs was directed towards the interfacial region, which induced major steric strain at the interface. Consequently, interaction of the protein with the hydrophobic domain of the amphiphile further disrupted its tertiary structure, which led to better opening up of the heme residue and, thereby, superior activity of the cyt c. The cyt c activity in the reverse micelles proportionately enhanced with an increase in the hydrophobicity of the GNP‐capping amphiphiles. A rigid cholesterol moiety as the hydrophobic end group of the GNP strikingly improved the cyt c activity by up to 200‐fold relative to that found in aqueous buffer. Fluorescence studies with both a tryptophan residue (Trp59) of the native protein and the sodium salt of fluorescein delineated the crucial role of the hydrophobicity of the GNP‐capping amphiphiles in improving the peroxidase activity of cyt c by unfolding its tertiary structure within the reverse micelles.     

Cancer Cell Imaging Using in Situ Generated Gold Nanoclusters

In situ generated fluorescent gold nanoclusters (Au‐NCs) are used for bio‐imaging of three human cancer cells, namely, lung (A549), breast (MCF7), and colon (HCT116), by confocal microscopy. The amount of Au‐NCs in non‐cancer cells (WI38 and MCF10A) is 20–40 times less than those in the corresponding cancer cells. The presence of a larger amount of glutathione (GSH) capped Au‐NCs in the cancer cell is ascribed to a higher glutathione level in cancer cells. The Au‐NCs exhibit fluorescence maxima at 490–530 nm inside the cancer cells. The fluorescence maxima and matrix‐assisted laser desorption ionization (MALDI) mass spectrometry suggest that the fluorescent Au‐NCs consist of GSH capped clusters with a core structure (Au_8‐13). Time‐resolved confocal microscopy indicates a nanosecond (1–3 ns) lifetime of the Au‐NCs inside the cells. This rules out the formation of aggregated Au–thiolate complexes, which typically exhibit microsecond (≈1000 ns) lifetimes. Fluorescence correlation spectroscopy (FCS) in live cells indicates that the size of the Au‐NCs is ≈1–2 nm. For in situ generation, we used a conjugate consisting of a room‐temperature ionic liquid (RTIL, [pmim][Br]) and HAuCl₄. Cytotoxicity studies indicate that the conjugate, [pmim][AuCl₄], is non‐toxic for both cancer and non‐cancer cells.     

Characterization of the Cytochrome c Membrane‐Binding Site Using Cardiolipin‐Containing Bicelles with NMR

Cytochrome (cyt) c transports electrons from Complex III to Complex IV in mitochondria. Cyt c is ordinarily anchored to the mitochondrial membrane through interaction with cardiolipin (CL), however its release into the cytosol initiates apoptosis. The cyt c interaction site with CL‐containing bicelles was characterized by NMR spectroscopy. Chemical shift perturbations in cyt c signals upon interaction with bicelles revealed that a relatively wide region, which includes the A‐site, the CXXCH motif, and the N‐ and C‐terminal helices, and contains multiple Lys residues, interacts cooperatively with CL. The specific cyt c–CL interaction increased with increasing CL molecules in the bicelles. The location of the cyt c interaction site for CL was similar to those for Complex III and Complex IV, thus indicating that cyt c recognizes lipids and partner proteins in a similar way. In addition to elucidating the cyt c membrane‐binding site, these results provide insight into the dynamic aspect of cyt c interactions in mitochondria.     

Reversible Switching of Redox‐Active Molecular Orbitals and Electron Transfer Pathways in CuA Sites of Cytochrome c Oxidase

The Cu_A site of cytochrome c oxidase is a redox hub that participates in rapid electron transfer at low driving forces with two redox cofactors in nearly perpendicular orientations. Spectroscopic and electrochemical characterizations performed on first and second‐sphere mutants have allowed us to experimentally detect the reversible switching between two alternative electronic states that confer different directionalities to the redox reaction. Specifically, the M160H variant of a native Cu_A shows a reversible pH transition that allows to functionally probe both states in the same protein species. Alternation between states exerts a dramatic impact on the kinetic redox parameters, thereby suggesting this effect as the mechanism underlying the efficiency and directionality of Cu_A electron transfer in vivo. These findings may also prove useful for the development of molecular electronics.     

Fluorescent Gold Nanoclusters with Interlocked Staples and a Fully Thiolate‐Bound Kernel

The structural features that render gold nanoclusters intrinsically fluorescent are currently not well understood. To address this issue, highly fluorescent gold nanoclusters have to be synthesized, and their structures must be determined. We herein report the synthesis of three fluorescent Au₂₄(SR)₂₀ nanoclusters (R=C₂H₄Ph, CH₂Ph, or CH₂C₆H₄^tBu). According to UV/Vis/NIR, differential pulse voltammetry (DPV), and X‐ray absorption fine structure (XAFS) analysis, these three nanoclusters adopt similar structures that feature a bi‐tetrahedral Au₈ kernel protected by four tetrameric Au₄(SR)₅ motifs. At least two structural features are responsible for the unusual fluorescence of the Au₂₄(SR)₂₀ nanoclusters: Two pairs of interlocked Au₄(SR)₅ staples reduce the vibration loss, and the interactions between the kernel and the thiolate motifs enhance electron transfer from the ligand to the kernel moiety through the Au?S bonds, thereby enhancing the fluorescence. This work provides some clarification of the structure–fluorescence relationship of such clusters.     

Gold Nanoclusters for Targeting Methicillin‐Resistant Staphylococcus aureus In Vivo

Widespread multidrug resistance caused by the abuse of antibiotics calls for novel strategies and materials. Gold nanoclusters (AuNCs) are scarcely explored for combating multidrug‐resistant (MDR) bacteria in vivo. We herein synthesized a novel class of AuNCs, namely quaternary ammonium (QA) capped AuNCs (QA‐AuNCs) as potent antibiotics selectively targeting MDR Gram‐positive bacteria, including methicillin‐resistant Staphylococcus aureus (MRSA) and vancomycin‐resistant Enterococci (VRE), in vivo. QA‐AuNCs kill bacteria through a combined physicochemical mechanism, and show excellent therapeutic effects in both a skin infection model and a bacteremia model induced by MRSA. In addition, owing to their intense fluorescence, QA‐AuNCs can be used for the discrimination of live/dead bacteria and bacteria counting, suggesting their potential for clinical theranostics.     

Cross‐β Amyloid Nanohybrids Loaded With Cytochrome C Exhibit Superactivity in Organic Solvents

The present study reports the development of a unique class of Cytochrome C (CytC)‐loaded cross‐beta amyloid nanohybrids. The peroxidase activity of the bound CytC increased up to two orders of magnitude in organic solvents compared to the activity of unbound CytC in water. The amyloid sequences used in the study feature the nucleating core ¹⁷LVFF²¹ of the beta amyloid (Aβ), which assembled to form homogenous fibers and nanotubes. The morphology and exposed surface of the amyloid nanohydrids critically modulated the CytC activity. A CytC–Ac‐KLVFFAE‐NH₂ hybrid featuring nanofiber morphology showed 308‐fold higher activity than unbound CytC in water, which increased to 450‐fold with the nanotube morphology of CytC–Ac‐KLVFFA L ‐NH₂. Notably, activity declined substantially when the exposed surface charge was detuned by replacing lysine with histidine, thus underpinning the importance of surface charge. This enzyme–amyloid nanohybrid system could facilitate the technological application of enzymes.     

Probing Viscosity of Co-Polymer Hydrogel and HeLa Cell Using Fluorescent Gold Nanoclusters: Fluorescence Correlation Spectroscopy and Anisotropy Decay

Fluorescence dynamics of gold nanoclusters (Au₉ and Au₂₅) are studied in the complex and crowded environment of a triblock co-polymer (F127) hydrogel and inside cervical cancer cell, HeLa. In the hydrogel, spherical micelles of F127 remain immobilized with a hydrophobic core (PPO) and a hydrophilic corona (PEO) region. The fluorescence anisotropy decay suggests that the timescale of rotational relaxation in the hydrogel is similar to that in bulk water (viscosity ∼1 cP). From fluorescence correlation spectroscopy (FCS) it is inferred that the local viscosity in the hydrogel is 12 cP for Au₉ and 18 cP for Au₂₃. These results indicate that gold nanoclusters (AuNCs) localize in the corona region of the hydrogel. Evidently, frictions against rotation and translation are different inside the gel. It is suggested that rotation of the AuNCs senses the immediate water-like “void” region while translation motion involves in-and-out movement of the AuNCs at the periphery of the gel. Finally, the gold nanoclusters are used for cell imaging and estimation of intracellular viscosity of HeLa cells.     

Cholesterol Hydroperoxides as Substrates for Cholesterol‐Metabolizing Cytochrome P450 Enzymes and Alternative Sources of 25‐Hydroxycholesterol and other Oxysterols

The interaction of the primary autoxidation products of cholesterol, namely 25‐ and 20ξ‐hydroperoxides, with the four principal cholesterol‐metabolizing cytochrome P450 enzymes is reported. Addition of cholesterol 25‐hydroperoxide to the enzymes CYP27A1 and CYP11A1 induced well‐defined spectral changes while generating 25‐hydroxycholesterol as the major product. The 20ξ‐hydroperoxides induced spectral shifts in CYP27A1 and CYP11A1 but glycol metabolites were detected only with CYP11A1. CYP7A1 and CYP46A1 failed to give metabolites with any of the hydroperoxides. A P450 hydroperoxide‐shunt reaction is proposed, where the hydroperoxides serve as both donor for reduced oxygen and substrate. CYP27A1 was shown to mediate the reduction of cholesterol 25‐hydroperoxide to 25‐hydroxycholesterol, a role of potential significance for cholesterol‐rich tissues with high oxidative stress. CYP27A1 may participate in the removal of harmful autoxidation products in these tissues, while providing a complementary source of 25‐hydroxycholesterol, a modulator of immune cell function and mediator of viral cell entry.     

Improved Cyclopropanation Activity of Histidine‐Ligated Cytochrome P450 Enables the Enantioselective Formal Synthesis of Levomilnacipran

Engineering enzymes capable of modes of activation unprecedented in nature will increase the range of industrially important molecules that can be synthesized through biocatalysis. However, low activity for a new function is often a limitation in adopting enzymes for preparative‐scale synthesis, reaction with demanding substrates, or when a natural substrate is also present. By mutating the proximal ligand and other key active‐site residues of the cytochrome P450 enzyme from Bacillus megaterium (P450‐BM3), a highly active His‐ligated variant of P450‐BM3 that can be employed for the enantioselective synthesis of the levomilnacipran core was engineered. This enzyme, BM3‐Hstar, catalyzes the cyclopropanation of N,N‐diethyl‐2‐phenylacrylamide with an estimated initial rate of over 1000 turnovers per minute and can be used under aerobic conditions. Cyclopropanation activity is highly dependent on the electronic properties of the P450 proximal ligand, which can be used to tune this non‐natural enzyme activity.     

Light Harvesting and White‐Light Generation in a Composite of Carbon Dots and Dye‐Encapsulated BSA‐Protein‐Capped Gold Nanoclusters

Several strategies have been adopted to design an artificial light‐harvesting system in which light energy is captured by peripheral chromophores and it is subsequently transferred to the core via energy transfer. A composite of carbon dots and dye‐encapsulated BSA‐protein‐capped gold nanoclusters (AuNCs) has been developed for efficient light harvesting and white light generation. Carbon dots (C‐dots) act as donor and AuNCs capped with BSA protein act as acceptor. Analysis reveals that energy transfer increases from 63 % to 83 % in presence of coumarin dye (C153), which enhances the cascade energy transfer from carbon dots to AuNCs. Bright white light emission with a quantum yield of 19 % under the 375 nm excitation wavelength is achieved by changing the ratio of components. Interesting findings reveal that the efficient energy transfer in carbon‐dot–metal‐cluster nanocomposites may open up new possibilities in designing artificial light harvesting systems for future applications.     

Redox‐State‐Mediated Regulation of Cytochrome c Release in Apoptosis Revealed by Surface‐Enhanced Raman Scattering on Nickel Substrates

The interaction of cytochrome c (Cyt c) with cardiolipin (CL) is believed to play an important role in the initial events of apoptosis. Herein, we investigate the structural changes of CL‐bound Fe²⁺Cyt c and the correlation with Cyt c release through surface‐enhanced Raman spectroscopy (SERS) on nickel substrates. The SERS results together with molecular dynamics simulation reveal that Fe²⁺Cyt c undergoes autoxidation and a relatively larger conformational alteration after binding with CL, inducing higher peroxidase activity of Cyt c and higher permeability of the CL membrane compared with those induced by the Fe³⁺Cyt c. The proapoptotic activity and SERS effect of the Ni nanostructures allow the in situ study of the redox‐state‐dependent Cyt c release from isolated mitochondria, which reveals for the first time that the ferrous state of Cyt c most likely plays a more important role in triggering apoptosis.     

Selective Killing of Breast Cancer Cells by Doxorubicin‐Loaded Fluorescent Gold Nanoclusters: Confocal Microscopy and FRET

Fluorescent gold nanoclusters (AuNCs) capped with lysozymes are used to deliver the anticancer drug doxorubicin to cancer and noncancer cells. Doxorubicin‐loaded AuNCs cause the highly selective and efficient killing (90 %) of breast cancer cells (MCF7) (IC₅₀=155 nm ). In contrast, the killing of the noncancer breast cells (MCF10A) by doxorubicin‐loaded AuNCs is only 40 % (IC₅₀=4500 nm ). By using a confocal microscope, the fluorescence spectrum and decay of the AuNCs were recorded inside the cell. The fluorescence maxima (at ≈490–515 nm) and lifetime (≈2 ns), of the AuNCs inside the cells correspond to Au_10–13. The intracellular release of doxorubicin from AuNCs is monitored by Förster resonance energy transfer (FRET) imaging.     

Silica‐Encapsulated Gd3+‐Aggregated Gold Nanoclusters for In Vitro and In Vivo Multimodal Cancer Imaging

Gd³⁺‐aggregated gold nanoclusters (AuNCs) encapsulated by silica shell (Gd³⁺‐A‐AuNCs@SiO₂ NPs) were strategically designed and prepared. The as‐prepared nanoparticles exhibit aggregation‐enhanced fluorescence (AEF), with an intensity that is up to 3.8 times that of discrete AuNCs. The clusters served as novel nanoprobes for in vitro and in vivo multimodal (fluorescence, magnetic resonance, and computed X‐ray tomography) cancer imaging     

Unraveling the Impact of Gold(I)–Thiolate Motifs on the Aggregation‐Induced Emission of Gold Nanoclusters

Aggregation‐induced emission (AIE) provides an efficient strategy to synthesize highly luminescent metal nanoclusters (NCs), however, rational control of emission energy and intensity of metal NCs is still challenging. This communication reveals the impact of surface Au^I‐thiolate motifs on the AIE properties of Au NCs, by employing a series of water‐soluble glutathione (GSH)‐coordinated Au complexes and NCs as a model ([Au₁₀SR₁₀], [Au₁₅SR₁₃], [Au₁₈SR₁₄], and [Au₂₅SR₁₈]^?, SR=thiolate ligand). Spectroscopic investigations show that the emission wavelength of Au NCs is adjustable from visible to the near‐infrared II (NIR‐II) region by controlling the length of the Au^I‐SR motifs on the NC surface. Decreasing the length of Au^I‐SR motifs also changes the origin of cluster luminescence from AIE‐type phosphorescence to Au⁰‐core‐dictated fluorescence. This effect becomes more prominent when the degree of aggregation of Au NCs increases in solution.     

An Unprecedented Kernel Growth Mode and Layer‐Number‐Odevity‐Dependent Properties in Gold Nanoclusters

Kernel atoms of Au nanoclusters are packed layer‐by‐layer along the [001] direction with every full (001) monolayer composed of 8 Au atoms (Au₈ unit) in nanoclusters with formula of Au_8n+4(TBBT)_4n+8 (n is the number of Au₈ units; TBBTH=4‐tert‐butylbenzenelthiol). It is unclear whether the kernel atoms can be stacked in a defective‐layer way along the [001] direction during growth of the series of nanoclusters and how the kernel layer number affects properties. Now, a nanocluster is synthesized that is precisely characterized by mass spectrometry and single‐crystal X‐ray crystallography, revealing a layer stacking mode in which a half monolayer composed of 4 atoms (Au₄ unit) is stacked on the full monolayer along the [001] direction. The size and the odevity of the kernel layer number influence the properties (polarity, photoluminescence) of gold nanoclusters. The obtained nanocluster extends the previous formula from Au_8n+4(TBBT)_4n+8 to Au_4n+4(TBBT)_2n+8 (n is the number of Au₄ units).