Session V41: Focus Session: Physics of Proteins I: Dynamics and Function

Two tyrosyl radicals stabilize high oxidation states in cytochrome c oxidase for efficient energy conservation and proton translocation

The reaction of hydrogen peroxide (H$_{2}$O$_{2})$ with oxidized bovine cytochrome $c$ oxidase (bC$c$O) was studied by electron paramagnetic resonance (EPR) to determine the properties of radical intermediates. Two distinct radicals with widths of 12 and 46 G are directly observed by X-band CW-EPR in the reaction of bC$c$O with H$_{2}$O$_{2}$ at pH 6 and pH 8. High-frequency EPR (D-band) provides assignments to tyrosine for both radicals based on well-resolved $g$-tensors. The 46 G wide radical has extensive hyperfine structure and can be fit with parameters consistent with Y129. However, the 12 G wide radical has minimal hyperfine structure and can be fit using parameters unique to the post-translationally modified Y244 in C$c$O. The results are supported by mixed quantum mechanics and molecular mechanics calculations. This study reports spectroscopic evidence of a radical formed on the modified tyrosine in C$c$O and resolves the much debated controversy of whether the wide radical seen at low pH in the bovine system is a tyrosine or tryptophan. A model is presented showing how radical formation and migration may play an essential role in proton translocation. This work was done in collaboration with Michelle A. Yu, Tsuyoshi Egawa, Syun-Ru Yeh and Gary J. Gerfen from Albert Einstein College of Medicine; Kyoko Shinzawa-Itoh and Shinya Yoshikawa from the University of Hyogo; and Victor Guallar from the Barcelona Supercomputing Center.   

Structural and electronic properties of copper-containing nitrite reductase (CuNiR): elucidating the mechanism of nitrite reduction at the T$_2$Cu center

Copper-containing nitrite reductases (CuNiRs) play an important role in catalyzing the reduction of NO$_2^{-}$ to NO during the bacterial denitrification process. Experimental studies have provided the structures of various states of CuNiR in the catalytic reaction, but many important aspects of the initial and intermediate attachments as well as the mechanism of the enzyme function remain unclear. We present a density-functional-theory-based study of the structural and electronic properties of different coordination forms at the T$_2$Cu center. The nudged elastic band (NEB) method is used to examine the activation energy barriers and to determine the minimum energy pathways (MEP) of the reaction processes. Our results reveal the role of the Asp$^{98}$ residue in the enzymatic function of CuNiR and also address the transformation from the initial O-coordinated binding of NO$_2^-$ to the N-coordinated attachment of the NO during the enzymatic reaction.   

Nanometer Scale Distance Measurements for Biological Systems using Gd$^{3+}$-based Spin Probes at High Magnetic Fields

Determination of nanometer-scale distances is critical for understanding structure and dynamics of proteins. Electron Paramagnetic Resonance (EPR), primarily below 1 T, is used to complement other structural techniques by quantifying sparse distances up to 8 nm in biomolecules labeled with nitroxide-based radicals. EPR becomes more powerful with increasing magnetic fields and frequencies. At 95 GHz (3.5 T), Gd$^{3+}$ ions have shown clear advantages over nitroxide probes (Potapov, JACS 2010). We show that these advantages are even more dramatic at 240 GHz (8.5 T). The width of Gd$^{3+}$'s central EPR transition narrows with increasing average distance between Gd$^{3+}$ ions out to distances as long as 5 nm. This doubles the distances accessible with nitroxides in continuous wave measurements, which can be carried out above the 200K protein-glass transition and with broad distance distributions. Temperature-dependent measurements of the phase memory times at 8.5 T and low temperatures show distance dependence out to 10 nm. Measurements of Gd$^{3+}$ labeled Proteorhodopsin confirm that phase memory times remain long enough to observe distance dependence in a spin-labeled protein. This work is supported by the National Science Foundation and the Binational Science Foundation.   

Metal-like transport in proteins: A new paradigm for biological electron transfer

Electron flow in biologically proteins generally occurs via tunneling or hopping and the possibility of electron delocalization has long been discounted. Here we report metal-like transport in protein nanofilaments, pili, of bacteria \textit{Geobacter sulfurreducens }that challenges this long-standing belief [1]. Pili exhibit conductivities comparable to synthetic organic metallic nanostructures. The temperature, magnetic field and gate-voltage dependence of pili conductivity is akin to that of quasi-1D disordered metals, suggesting a metal-insulator transition. Magnetoresistance (MR) data provide evidence for quantum interference and weak localization at room temperature, as well as a temperature and field-induced crossover from negative to positive MR. Furthermore, pili can be doped with protons. Structural studies suggest the possibility of molecular pi stacking in pili, causing electron delocalization. Reducing the disorder increases the metallic nature of pili. These electronically functional proteins are a new class of electrically conductive biological proteins that can be used to generate future generation of inexpensive and environmentally-sustainable nanomaterials and nanolectronic devices such as transistors and supercapacitors. [1] Malvankar et al. Nature Nanotechnology, 6, 573-579 (2011)   

Behind the Scene Role of Conserved Threonine in Intein Splicing

Protein splicing is an autocatalytic process where an ``intein'' self-cleaves from a precursor protein and catalyzes ligation of the flanking fragments. Inteins occur in all domains of life and have myriad uses in biotechnology. While reaction steps of intein splicing are known, mechanistic details remain incomplete. Here, we investigate the possible role of a highly conserved active-site Threonine residue in bringing about the initial step of splicing: peptide bond rearrangement at a conserved Glycine-Cysteine motif. We report that although not part of the active transition state in this reaction, Threonine plays an important role in reducing the energy barrier through charge screening of active residues in the transition state. Interestingly, Threonine-Glycine hydrogen bonding makes sulfur of the attacking Cysteine less nucleophilic, thereby minimizing Coulomb repulsion in the transition state. These non-intuitive results are obtained through a combination of crystal structure, quantum mechanical simulations, and mutagenesis data. Our results further predict that the sluggish reaction rates observed with intein mutants harboring Threonine-Alanine substitutions can be accelerated in the presence of non-aqueous solvents.   

Coherent Oscillations in Hemoglobin and Myoglobin Ligand Complexes and their Dynamical Connection to Global Protein Motion and Gas Discrimination

THz spectroscopy and Molecular Dynamics (MD) simulations are used to investigate the coherent oscillations in the heme group of two heme proteins (hemoglobin and myoglobin) and the affect of the heme dynamics on the collective fluctuations taking place in the proteins. Preliminary experiments have confirmed that the deoxy state (no gas in the active site) of both proteins do not possess a 40 cm$^{-1}$ mode in their THz spectra. The 40 cm$^{-1 }$mode has been observed in both experimental and theoretical investigations of myoglobin dynamics. The low-frequency mode at 40 cm$^{-1}$ has been hypothesized to be connected with energy transport between the active site and the protein-solvent interface. Once a gas is introduced into the ligand, both proteins contain the 40 cm$^{-1}$ mode in their experimental spectra. But both the shape and intensity of the myoglobin peak differs from that of hemoglobin. Additionally, we observe a number of collective protein fluctuations ($\le $ 100 cm$^{-1})$ that are altered in the myoglobin spectrum but remain unchanged in the hemoglobin spectrum when a gas is introduced into the protein active site. We will present experimental data of both proteins that have been exposed to a number of different gases. The reaction of the protein collective motions in the gas is linked with the difference in the coupling of the coherent oscillations of the heme group with the protein global modes but also with the mechanism of protein relaxation that controls ligand migration.   

Vibrational Dynamics of Ferric MbCN-A Revisit by Resonance Raman and Vibrational Coherence Spectroscopy

Ultrafast pump-probe spectroscopy has indicated that there exists a photoproduct state following the excitation of ferric MbCN$^{[1][2]}$. This excited state decays with a time constant of 3.6 ps$^{[1]}$. Previous studies on this system have suggested that in this photoproduct state, the heme is either (i) still six-coordinated but vibrationally hot in the electronic ground state$^{[1]}$ or (ii) the proximal histidine residue (His93) is transiently dissociated, while CN$^{-}$ is still bound$^{[2]}$. Recent resonance Raman measurements on ferric MbCN in static solution yield spectra that are very similar to ferric myoglobin, which has His93 and a water molecule as axial ligands. This indicates that a water molecule replaces CN$^{-}$ in ferric MbCN under continuous laser excitation. Photolysis of CN$^{-}$ from the heme iron is necessary to make this happen, which is not consistent with the above two suggestions. In this presentation we will revisit the dynamics of ferric MbCN with resonance Raman and vibrational coherence spectroscopy and try to explain how a water molecule competes with CN$^{-}$ in binding to the heme under photo excitation$^{[3]}$. References: [1]Helbing J. et al., Biophys J, vol 87, 1881(2004) [2]Gruia F. et al., Biophys J, vol 94, 2252(2008) [3]Cao W. et al., Biochemistry, vol 40, 5728(2001)   

Effect of DNA binding on geminate CO recombination kinetics in CooA

CooA proteins are heme-based CO-sensing transcription factors. Here we study the ultrafast dynamics of geminate CO rebinding to RrCooA. The effects of DNA binding and the truncation of the DNA binding domain on the CO geminate recombination kinetics were investigated. The CO rebinding kinetics in these CooA complexes takes place on ultrafast timescales but remains non-exponential over many decades in time. We show that this non-exponential kinetic response is due to a quenched enthalpic barrier distribution resulting from a distribution of heme geometries that is frozen or slowly evolving on the timescale of CO rebinding. We also show that, upon CO binding, the distal pocket of the heme in RrCooA relaxes to form a very efficient hydrophobic trap for CO. DNA binding further tightens the narrow distal pocket and slightly weakens the iron-proximal histidine bond. Analysis of our data reveals that the uncomplexed and inherently flexible DNA binding domain adds additional structural heterogeneity to the heme doming coordinate. When CooA forms a complex with DNA, the flexibility of the DNA-binding domain decreases and the distribution of the conformations available in the heme domain becomes restricted.   

Physical control of carrier-mediated ion-transporters by entrainment of their turnover rate

In the past, tremendous efforts have been made to physically activate carrier-mediated ion-transporters, such as Na/K pumps. However, the outcome is not significant. Recently, we developed a new technique which can effectively and efficiently control the pumping rate by introducing a concept of an electronic synchrotron accelerator to the biological system. The approach consists of two steps. First, a specially designed oscillating electric field is used to force or synchronize individual pump molecules to run at the same turnover rate and phase as the field oscillation frequency. Then, by gradually changing the field frequency and carefully keeping the pump synchronization we can entrain the pump molecules so that their pumping rate can be progressively modulated, either decelerated or accelerated, following the field frequency to a defined value. Based on theoretical analysis of the underlying mechanisms involved in the technique, computer simulation of the entrainment process, and intensive experimental studies we have realized significant activation of the Na/K pumping rate up to ten-folds quickly in less than ten seconds.   

Molecular dynamics simulation studies of dielectric response and vibrational energy relaxation in photoactive yellow protein and green fluorescent protein

The first step in the photocycle of many proteins involves conformational change of a chromophore or a charge transfer reaction following photoexcitation. To explore the response of the protein and solvent environment to photoexcitation of the chromophore in photoactive yellow protein (PYP) and green fluorescent protein (GFP) we carried out molecular dynamics simulations of the dielectric response and vibrational energy relaxation (VER) from the chromophore to the protein and solvent. In PYP the time scale of the protein response, mainly contributed by Tyr42 and Glu46, to photoexcitation appears prominently between 0.1 and 0.3 picoseconds. The frequency-dependent VER rate also reveals dynamic coupling between the chromophore and residues that hydrogen bond to it. Resonances in the VER rate appear at frequencies comparable to the oscillations observed in recent fluorescence decay studies. In GFP, which undergoes excited state proton transfer about 10 ps following photoexcitation that may be assisted by specific chromophore vibrations, both the protein and water molecules inside the $\beta $-barrel surrounding the chromophore mediate the dielectric response.   

Employing Solution X-Ray Scattering to Investigate Conformational Changes of Proteins

Many proteins undergo global conformational changes to perform their functions. Small/Wide Angle Solution X-Ray Scattering (S/WAXS) is an increasingly used experimental technique to probe conformational changes of proteins in aqueous environment. We have developed a new modeling method to reconstruct coarse-grained and atomistic 3D models from S/WAXS data. The method has been applied to a variety of proteins including the R-T transition of hemoglobin.   

Near-Field Orientation Sensitive Terahertz Micro-Spectroscopy of Single Crystals

We present spectroscopic imaging studies of molecular crystals. These measurements examine the anisotropy of the intra and inter-molecular vibrational modes of single crystals at terahertz frequencies. The method is based on the technique developed in [1-2] for sub-wavelength resolution time domain terahertz spectroscopy (THz TDS), with added polarization orientation dependent measurements and hydration control. This method allows us to study the spectroscopic properties of small single crystals with sizes down to 20 micrometers. In addition, mapping the spectroscopic information at such small spatial scales allows us to reduce the water absorption and interference artifacts that usually affect protein THz TDS measurements. We show the polarization sensitive terahertz absorption spectra in the (0.3-3THz) range of sucrose, oxalic acid and lysozyme protein crystals. \begin{enumerate} \item M. A. Seo, et. al., Opt. Express, 15(19):11781--11789, 09 (2007) \item J. R Knab, et. al., App. Phys. Lett.,97, 031115 (2010) \end{enumerate}