R. Brian Dyer

Bound water in the proton translocation mechanism of the haem-copper oxidases

We address the molecular mechanism by which the haem‐copper oxidases translocate protons. Reduction of O2 to water takes place at a haem iron‐copper (CuB) centre, and protons enter from one side of the membrane through a ‘channel’ structure in the enzyme. Statistical‐mechanical calculations predict bound water molecules within this channel, and mutagenesis experiments show that breaking this water structure impedes proton translocation. Hydrogen‐bonded water molecules connect the channel further via a conserved glutamic acid residue to a histidine ligand of CuB. The glutamic acid side chain may have to move during proton transfer because proton translocation is abolished if it is forced to interact with a nearby lysine or arginine. Perturbing the CuB ligand structure shifts an infrared mode that may be ascribed to the OH stretch of bound water. This is sensitive to mutations of the glutamic acid, supporting its connectivity to the histidine. These results suggest key roles of bound water, the glutamic acid and the histidine copper ligand in the mechanism of proton translocation.

Azidohomoalanine: A Conformationally Sensitive IR Probe of Protein Folding, Protein Structure, and Electrostatics

IR probes are widely used to study protein dynamics,[1] protein–ligand interactions, and electric field effects in proteins, but such probes can suffer from poor sensitivity or can be difficult to introduce.[1a,f,2] The approach requires the introduction of an IR active reporter group into the protein of interest. Here we show that an azido-bearing nonnatural amino acid, azidohomoalanine (Aha), provides a high-sensitivity probe of protein structure, protein folding, and protein electrostatics. Interest in the azido group was initially driven by its application in bioorthogonal protein labeling and click chemistry.[3a] Use of the azido group as an IR probe has a number of attractive features. Its absorbance falls in an otherwise transparent region of the IR spectrum and the frequency is sensitive to the environment.[4] Of particular importance, the extinction coefficient of an azido group is approximately twenty-fold larger than that of the commonly employed cyano group.[4a] Azidohomoalanine (Aha) can be viewed as an analogue of Met and, thus, one can realize its incorporation using well developed Met auxotrophic strains (Figure 1a).[3] The group is also compatible with the conditions of Fmoc solid-phase peptide synthesis. Figure 1 a) Structure of azidohomoalanine (Aha) and methionine (Met). b) A ribbon diagram of NTL9 showing the location of Met1 (pink) and Ile 4 (orange). The diagram was generated using the pdb file 2HBA and the program Pymol. We used the N-terminal domain of the ribosomal protein L9 (NTL9) as a test case. NTL9 is a 56-residue protein, with a mixed β–α structure that has been studied extensively as a model system for protein folding and stability. It folds cooperatively under a wide range of conditions.[5] We targeted Met1 by expression (NTL9-Met1Aha) and Ile4 (NTL9*-Ile4Aha) by chemical synthesis as sites for incorporation of the Aha substitutions (Figure 1b). NTL9* refers to a K12A mutant of NTL9 which adopts the same fold, but is more stable than the wild-type. Both of these positions are in the hydrophobic core of the protein (Figure 1b). Azidohomoalanine was synthesized in four steps starting from homoserine in 17% overall yield (Supporting Information). Protein expression was performed with the standard methionine auxotrophic E. coli strain, B834. Mass spectrometry was used to test the level of incorporation. The experimental and theoretical mass spectra of NTL9-Met1Aha match extremely well and a minimum incorporation of 94% was detected by MS–MS analysis (Supporting Information). The small amount of wild-type protein is spectroscopically silent in the azido vibration region of the IR spectrum. Spectroscopic analysis shows that the substitution does not perturb the structure of the protein. The far-UV CD spectrum of the M1Aha mutant is similar to the spectrum of the wild-type indicating that they have similar secondary structure. The 1D-NMR spectrum of the mutant displays the characteristic ring current shifted methyl resonances of NTL9, as well as Cα proton chemical shifts downfield of H2O, indicative of β-sheet structure, and the NOE spectrum shows that the native registry of the β-sheet is maintained (Supporting Information). The potential effects of the substitution on the stability and cooperativity of folding were also examined. Urea denaturation shows a sigmoidal curve, as expected for a cooperatively folded unit. The stability is decreased relative to the wild-type by 0.81 kcalmol−1. The m-value, which is the slope of the ΔG° vs [urea] curve, is very similar to that of the wild-type. m-values are widely believed to be related to the change in accessible surface area between the folded and unfolded states and the good agreement between wild-type and mutant provides additional evidence of the structural integrity of the mutant.[6] Thermal unfolding is also cooperative and the melting point (Tm) is decreased by 5 to 6°C (Supporting Information). Stopped-flow kinetic refolding studies confirm that folding is two-state (Supporting Information). IR spectra were recorded in the folded state and in the unfolded state induced by temperature. The folded-state spectrum was recorded at pD 8.8 to ensure a single protonation state for the N-terminus since a partially protonated N-terminus might lead to a splitting of the folded-state peak due to electrostatic effects. NTL9 is fully folded at pD 8.8, 20°C (Supporting Information). In the folded state, the azido band is at 2094 cm−1. In the unfolded state at 90°C, a single broad band is found at 2113 cm−1, indicating exposure of the azido group to water (Figure 2). The significant band shift observed between the folded and unfolded states indicates that the azido vibration can be used as a probe of protein folding and hydrophobic burial of side chain. Figure 2 FTIR spectra of a) NTL9-Met1Aha and b) NTL9*-Ile4Aha in the folded state at 20°C (continuous line) and in the unfolded state at 90°C (broken line). The methodology is not limited to selective incorporation of a Met at the N-terminus since methionyl aminopeptidase has been shown to cleave azidohomoalanine from proteins expressed in E. coli depending on the solvent exposure of Met1 and the amino acid following Met1. Methionyl amino-peptidase cleaves N-terminal azidohomoalanine if the next amino acid is small.[3b] In NTL9, Met1 is part of the hydrophobic core and is retained when the protein is expressed in normal cell lines. We demonstrated that the probe can also be incorporated into proteins by solid-phase peptide synthesis. Ile4 in NTL9 was replaced by Aha using Fmoc based methods (Supporting Information). The Ile4Aha variant adopted the same fold as wild-type as judged by CD, 1D, and 2D-NMR spectroscopy. Thermal and denaturant induced unfolding are still cooperative and stopped-flow kinetic studies confirm that folding is two-state (Supporting Information). Substitution of Aha for Ile4 is more destabilizing than for Met1 with a ΔΔG° of 1.9 kcal mol−1 (Supporting Information). The larger effect is likely due to the fact that Aha is approximately isosteric for Met but not for Ile. IR spectra of the Ile4Aha mutant were taken in the folded state and in the thermally unfolded state. In the folded state the azido vibration was observed at 2105 cm−1 and in the unfolded state the vibration blue-shifts to 2112 cm−1 (Figure 2). The observed frequency for Aha in the folded state of the Ile4Aha mutant at 20°C indicates that azido group is more exposed to solvent than in the Met1Aha mutant. Ile4 packs against the C-terminal helix and this helix is known to fray at its C-terminus in the wild-type. The change in shape and polarity of the substitution may enhance this effect in the mutant and account for the partial solvent exposure. Irrespective of the details, the experiments with Ile4Aha demonstrate that Aha can be incorporated by solid-phase peptide synthesis and reiterate that the IR vibration of the azido group is sensitive to its environment. Spectra were also recorded of the Met1Aha variant at pD 6.0 to test the sensitivity of the probe to varying nearby charges. NTL9 is fully folded under these conditions but the N-terminus is partially protonated. Figure 3 compares the spectrum recorded at pD 6.0 to that measured at pD 8.8. The pD 6.0 spectrum displays a second partially resolved peak at 2116 cm−1 which is due to the protonated form (Supporting Information) and a major peak at 2094 cm−1. The drastic change observed upon partial protonation of the N-terminus demonstrates the sensitivity of the azido group vibrational mode to changes in electrostatic effects and minor native state structural variations. Figure 3 FTIR spectra of the folded state of NTL9-Met1Aha at pD 6.0 (broken line) and pD 8.8 (continuous line). We have demonstrated that azidohomoalanine is a sensitive IR probe of protein folding, protein structure, and electrostatic effects. The probe can be easily incorporated into proteins in high yield in a site-specific manner using simple, readily available auxotrophic expression systems as well as by solid-phase peptide synthesis. The probe provides a complimentary approach to methods involving orthogonal amino-acyl tRNA synthetases or approaches that involve the attachment of probes to introduced cysteine residues and should be generally accessible.[1d,2c]

The helix–turn–helix motif as an ultrafast independently folding domain: The pathway of folding of Engrailed homeodomain

Helices 2 and 3 of Engrailed homeodomain (EnHD) form a helix–turn–helix (HTH) motif. This common motif is believed not to fold independently, which is the characteristic feature of a motif rather than a domain. But we found that the EnHD HTH motif is monomeric and folded in solution, having essentially the same structure as in full-length protein. It had a sigmoidal thermal denaturation transition. Both native backbone and local tertiary interactions were formed concurrently at 4 × 105 s−1 at 25°C, monitored by IR and fluorescence T-jump kinetics, respectively, the same rate constant as for the fast phase in the folding of EnHD. The HTH motif, thus, is an ultrafast-folding, natural protein domain. Its independent stability and appropriate folding kinetics account for the stepwise folding of EnHD, satisfy fully the criteria for an on-pathway intermediate, and explain the changes in mechanism of folding across the homeodomain family. Experiments on mutated and engineered fragments of the parent protein with different probes allowed the assignment of the observed kinetic phases to specific events to show that EnHD is not an example of one-state downhill folding.

Direct Evidence of Active-Site Reduction and Photodriven Catalysis in Sensitized Hydrogenase Assemblies

We report photocatalytic H(2) production by hydrogenase (H(2)ase)-quantum dot (QD) hybrid assemblies. Quenching of the CdTe exciton emission was observed, consistent with electron transfer from the quantum dot to H(2)ase. GC analysis showed light-driven H(2) production in the presence of a sacrificial electron donor with an efficiency of 4%, which is likely a lower limit for these hybrid systems. FTIR spectroscopy was employed for direct observation of active-site reduction in unprecedented detail for photodriven H(2)ase catalysis with sensitivity toward both H(2)ase and the sacrificial electron donor. Photosensitization with Ru(bpy)(3)(2+) showed distinct FTIR photoreduction properties, generating all of the states along the steady-state catalytic cycle with minimal H(2) production, indicating slow, sequential one-electron reduction steps. Comparing the H(2)ase activity and FTIR results for the two systems showed that QDs bind more efficiently for electron transfer and that the final enzyme state is different for the two sensitizers. The possible origins of these differences and their implications for the enzymatic mechanism are discussed.

Probing protein dynamics using temperature jump relaxation spectroscopy

There have been recent advances in initiating and perturbing chemical reactions on very fast timescales, as short as picoseconds, thus making it feasible to study a vast range of chemical kinetics problems that heretofore could not be studied. One such approach is the rapid heating of water solutions using laser excitation. Laser-induced temperature jump relaxation spectroscopy can be used to determine the dynamics of protein motion, an area largely unstudied for want of suitable experimental and theoretical probes, despite the obvious importance of dynamics to protein function. Coupled with suitable spectroscopic probes of structure, relaxation spectroscopy can follow the motion of protein atoms over an enormous time range, from picoseconds to minutes (or longer), and with substantial structural specificity.

Nanosecond temperature jump relaxation dynamics of cyclic β-hairpin peptides

The thermal unfolding of a series of 6-, 10-, and 14-mer cyclic β-hairpin peptides was studied to gain insight into the mechanism of formation of this important secondary structure. The thermodynamics of the transition were characterized using temperature dependent Fourier transform infrared spectroscopy. Thermodynamic data were analyzed using a two-state model which indicates increasing cooperativity along the series. The relaxation kinetics of the peptides in response to a laser induced temperature jump were probed using time-resolved infrared spectroscopy. Single exponential relaxation kinetics were observed and fit with a two-state model. The folding rate determined for these cyclic peptides is accelerated by some two orders of magnitude over the rate of a linear peptide that forms a β-hairpin. This observation supports the argument that the rate limiting step in the linear system is either stabilization of compact collapsed structures or rearrangement of collapsed structures over a barrier to achieve the native interstrand registry. Small activation energies for folding of these peptides obtained from an Arrhenius analysis of the rates imply a primarily entropic barrier, hence an organized transition state having specific stabilizing interactions.

Resonance Raman studies of Rieske-type proteins

Resonance Raman (RR) spectra are reported for the [2Fe-2S] Rieske protein from Thermus thermophilus (TRP) and phthalate dioxygenase from Pseudomonas cepacia (PDO) as a function of pH and excitation wavelength. Depolarization ratio measurements are presented for the RR spectra of spinach ferredoxin (SFD), TRP, and PDO at 74 K. By comparison with previously published RR spectra of SFD, we suggest reasonable assignments for the spectra of TRP and PDO. The spectra of PDO exhibit virtually no pH dependence, while significant changes are observed in TRP spectra upon raising the pH from 7.3 to 10.1. One band near 270 cm-1, which consists of components at 266 cm-1 and 274 cm-1, is attributed to Fe(III)-N(His) stretching motions. We suggest that these two components arise from conformers having a protonated-hydrogen-bonded imidazole (266 cm-1) and deprotonated-hydrogen-bonded imidazolate (274 cm-1) coordinated to the Fe/S cluster and that the relative populations of the two species are pH-dependent; a simple structural model is proposed to account for this behavior in the respiratory-type Rieske proteins. In addition, we have identified RR peaks associated with the bridging and terminal sulfur atoms of the Fe-S-N cluster. The RR excitation profiles of peaks associated with these atoms are indistinguishable from each other in TRP (pH 7.3) and PDO and differ greatly from those of [2Fe-2S] ferrodoxins. The profiles are bimodal with maxima near 490 nm and > approx. 550 nm. By contrast, bands associated with the Fe-N stretch show a somewhat different enhancement profile. Upon reduction, RR peaks assigned to Fe-N vibrations are no longer observed, with the resulting spectrum being remarkably similar to that reported for reduced adrenodoxin. This indicates that only modes associated with Fe-S bonds are observed and supports the idea that the reducing electron resides on the iron atom coordinated to the two histidine residues. Taken as a whole, the data are consistent with an St2FeSb2Fe[N(His)]t2 structure for the Rieske-type cluster.

Proton-coupled electron transfer dynamics in the catalytic mechanism of a [NiFe]-hydrogenase.

The movement of protons and electrons is common to the synthesis of all chemical fuels such as H2. Hydrogenases, which catalyze the reversible reduction of protons, necessitate transport and reactivity between protons and electrons, but a detailed mechanism has thus far been elusive. Here, we use a phototriggered chemical potential jump method to rapidly initiate the proton reduction activity of a [NiFe] hydrogenase. Coupling the photochemical initiation approach to nanosecond transient infrared and visible absorbance spectroscopy afforded direct observation of interfacial electron transfer and active site chemistry. Tuning of intramolecular proton transport by pH and isotopic substitution revealed distinct concerted and stepwise proton-coupled electron transfer mechanisms in catalysis. The observed heterogeneity in the two sequential proton-associated reduction processes suggests a highly engineered protein environment modulating catalysis and implicates three new reaction intermediates; Nia-I, Nia-D, and Nia-SR(-). The results establish an elementary mechanistic understanding of catalysis in a [NiFe] hydrogenase with implications in enzymatic proton-coupled electron transfer and biomimetic catalyst design.

Anisotropic energy flow and allosteric ligand binding in albumin

Allosteric interactions in proteins generally involve propagation of local structural changes through the protein to a remote site. Anisotropic energy transport is thought to couple the remote sites, but the nature of this process is poorly understood. Here, we report the relationship between energy flow through the structure of bovine serum albumin and allosteric interactions between remote ligand binding sites of the protein. Ultrafast infrared spectroscopy is used to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic and anisotropic energy flow through the protein structure following input of thermal energy into the flexible ligand binding sites, without local heating of the rigid helix bundles that connect these sites. This efficient energy transport mechanism enables the allosteric propagation of binding energy through the connecting helix structures.

CO2 reduction catalyzed by mercaptopteridine on glassy carbon.

The catalytic reduction of CO2 is of great current interest because of its role in climate change and the energy cycle. We report a pterin electrocatalyst, 6,7-dimethyl-4-hydroxy-2-mercaptopteridine (PTE), that catalyzes the reduction of CO2 and formic acid on a glassy carbon electrode. Pterins are natural cofactors for a wide range of enzymes, functioning as redox mediators and C1 carriers, but they have not been exploited as electrocatalysts. Bulk electrolysis of a saturated CO2 solution in the presence of the PTE catalyst produces methanol, as confirmed by gas chromatography and (13)C NMR spectroscopy, with a Faradaic efficiency of 10-23%. FTIR spectroelectrochemistry detected a progression of two-electron reduction products during bulk electrolysis, including formate, aqueous formaldehyde, and methanol. A transient intermediate was also detected by FTIR and tentatively assigned as a PTE carbamate. The results demonstrate that PTE catalyzes the reduction of CO2 at low overpotential and without the involvement of any metal.