Adam R. Offenbacher

pH-Dependent cis --> trans isomerization rates for azobenzene dyes in aqueous solution.

Azobenzenes can function as molecular switches driven by their unusual cis <--> trans photoisomerization properties. The stability of an azobenzene-based switch depends on its rate of thermal relaxation, which is known to depend on the solvent environment, but few kinetic studies in aqueous media have been reported. We use nanosecond UV laser flash photolysis-transient absorption spectroscopy to measure thermal cis --> trans isomerization rates for mono- and disubstituted p-aminoazobenzenes and p-hydroxyazobenzenes in water at 23 degrees C over the pH range of 4 to 11. Observed absorption transients are fit to first-order relaxation rate constants between 10(5) and 10(1) s(-1), which is generally much faster than in nonpolar solvents, and the relaxation rates vary systematically and predictably with pH as the equilibrium shifts to ionized forms of the dyes that isomerize much more rapidly. Acid ionization constants for these dyes determined from our kinetic mechanism are compared with the pH dependence of their equilibrium UV-vis spectra. New kinetics results may enable pH control of azobenzene-based molecular switching times.

Redox-Linked Conformational Control of Proton Coupled Electron Transfer: Y122 in the Ribonucleotide Reductase β2 Subunit

Tyrosyl radicals play essential roles in biological proton-coupled electron transfer (PCET) reactions. Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides and is vital in DNA replication in all organisms. Class Ia RNRs consist of α2 and β2 homodimeric subunits. In class Ia RNR, such as the E. coli enzyme, an essential tyrosyl radical (Y122O(•))-diferric cofactor is located in β2. Although Y122O(•) is extremely stable in free β2, Y122O(•) is highly reactive in the quaternary substrate-α2β2 complex and serves as a radical initiator in catalytic PCET between β2 and α2. In this report, we investigate the structural interactions that control the reactivity of Y122O(•) in a model system, isolated E. coli β2. Y122O(•) was reduced with hydroxyurea (HU), a radical scavenger that quenches the radical in a clinically relevant reaction. In the difference FT-IR spectrum, associated with this PCET reaction, amide I (CO) and amide II (CN/NH) bands were observed. Specific (13)C-labeling of the tyrosine C1 carbon assigned a component of these bands to the Y122-T123 amide bond. Comparison to density functional calculations on a model dipeptide, tyrosine-threonine, and structural modeling demonstrated that PCET is associated with a Y122 rotation and a 7.2 Å translation of the Y122 phenolic oxygen. To test for the functional consequences of this structural change, a proton inventory defined the origin of the large solvent isotope effect (SIE = 16.7 ± 1.0 at 25 °C) on this reaction. These data suggest that the one-electron, HU-mediated reduction of Y122O(•) is associated with two, rate-limiting (full or partial) proton transfer reactions. One is attributable to HU oxidation (SIE = 11.9, net H atom transfer), and the other is attributable to coupled, hydrogen-bonding changes in the Y122O(•)-diferric cofactor (SIE = 1.4). These results illustrate the importance of redox-linked changes to backbone and ring dihedral angles in high potential PCET and provide evidence for rate-limiting, redox-linked hydrogen-bonding interactions between Y122O(•) and the iron cluster.

An Intrinsically Disordered Photosystem II Subunit, PsbO, Provides a Structural Template and a Sensor of the Hydrogen-Bonding Network in Photosynthetic Water Oxidation

Background: PsbO is an intrinsically disordered subunit of photosystem II. Results: Temperature-sensitive PsbO dynamics are identified by reaction-induced Fourier transform infrared spectroscopy and the removal and reconstitution of PsbO. Conclusion: PsbO serves as an organizational template and undergoes flash-induced hydrogen-bonding changes, coupled with the catalytic cycle of water oxidation. Significance: PsbO samples a rough conformational landscape when bound to its target, the photosystem II reaction center. Photosystem II (PSII) is a membrane-bound enzyme that utilizes solar energy to catalyze the photooxidation of water. Molecular oxygen is evolved after four sequential light-driven oxidation reactions at the Mn4CaO5 oxygen-evolving complex, producing five sequentially oxidized states, Sn. PSII is composed of 17 membrane-spanning subunits and three extrinsic subunits, PsbP, PsbQ, and PsbO. PsbO is intrinsically disordered and plays a role in facilitation of the water oxidizing cycle. Native PsbO can be removed and substituted with recombinant PsbO, thereby restoring steady-state activity. In this report, we used reaction-induced Fourier transform infrared spectroscopy to obtain information concerning the role of PsbP, PsbQ, and PsbO during the S state cycle. Light-minus-dark difference spectra were acquired, monitoring structural changes associated with each accessible flash-induced S state transition in a highly purified plant PSII preparation (Triton X-100, octylthioglucoside). A comparison of S2 minus S1 spectra revealed that removal of PsbP and PsbQ had no significant effect on the data, whereas amide frequency and intensity changes were associated with PsbO removal. These data suggest that PsbO acts as an organizational template for the PSII reaction center. To identify any coupled conformational changes arising directly from PsbO, global 13C-PsbO isotope editing was employed. The reaction-induced Fourier transform infrared spectra of accessible S states provide evidence that PsbO spectral contributions are temperature (263 and 277 K) and S state dependent. These experiments show that PsbO undergoes catalytically relevant structural dynamics, which are coupled over long distance to hydrogen-bonding changes at the Mn4CaO5 cluster.

Perturbations of aromatic amino acids are associated with iron cluster assembly in ribonucleotide reductase.

The β2 subunit of class Ia ribonucleotide reductases (RNR) contains an antiferromagnetically coupled μ-oxo bridged diiron cluster and a tyrosyl radical (Y122•). In this study, an ultraviolet resonance Raman (UVRR) difference technique describes the structural changes induced by the assembly of the iron cluster and by the reduction of the tyrosyl radical. Spectral contributions from aromatic amino acids are observed through UV resonance enhancement at 229 nm. Vibrational bands are assigned by comparison to histidine, phenylalanine, tyrosine, tryptophan, and 3-methylindole model compound data and by isotopic labeling of histidine in the β2 subunit. Reduction of the tyrosyl radical reveals Y122• Raman bands at 1499 and 1556 cm(-1) and Y122 Raman bands at 1170, 1199, and 1608 cm(-1). There is little perturbation of other aromatic amino acids when Y122• is reduced. Assembly of the iron cluster is shown to be accompanied by deprotonation of histidine. A p(2)H titration study supports the assignment of an elevated pK for the histidine. In addition, structural perturbations of tyrosine and tryptophan are detected. For tryptophan, comparison to model compound data suggests an increase in hydrogen bonding and a change in conformation when the iron cluster is removed. pH and (2)H(2)O studies imply that the perturbed tryptophan is in a low dielectric environment that is close to the metal center and protected from solvent exchange. Tyrosine contributions are attributed to a conformational or hydrogen-bonding change. In summary, our work shows that electrostatic and conformational perturbations of aromatic amino acids are associated with metal cluster assembly in RNR. These conformational changes may contribute to the allosteric effects, which regulate metal binding.

13C ENDOR Spectroscopy of Lipoxygenase–Substrate Complexes Reveals the Structural Basis for C–H Activation by Tunneling

In enzymatic C–H activation by hydrogen tunneling, reduced barrier width is important for efficient hydrogen wave function overlap during catalysis. For native enzymes displaying nonadiabatic tunneling, the dominant reactive hydrogen donor–acceptor distance (DAD) is typically ca. 2.7 Å, considerably shorter than normal van der Waals distances. Without a ground state substrate-bound structure for the prototypical nonadiabatic tunneling system, soybean lipoxygenase (SLO), it has remained unclear whether the requisite close tunneling distance occurs through an unusual ground state active site arrangement or by thermally sampling conformational substates. Herein, we introduce Mn2+ as a spin-probe surrogate for the SLO Fe ion; X-ray diffraction shows Mn-SLO is structurally faithful to the native enzyme. 13C ENDOR then reveals the locations of 13C10 and reactive 13C11 of linoleic acid relative to the metal; 1H ENDOR and molecular dynamics simulations of the fully solvated SLO model using ENDOR-derived restraints give additional metrical information. The resulting three-dimensional representation of the SLO active site ground state contains a reactive (a) conformer with hydrogen DAD of ∼3.1 Å, approximately van der Waals contact, plus an inactive (b) conformer with even longer DAD, establishing that stochastic conformational sampling is required to achieve reactive tunneling geometries. Tunneling-impaired SLO variants show increased DADs and variations in substrate positioning and rigidity, confirming previous kinetic and theoretical predictions of such behavior. Overall, this investigation highlights the (i) predictive power of nonadiabatic quantum treatments of proton-coupled electron transfer in SLO and (ii) sensitivity of ENDOR probes to test, detect, and corroborate kinetically predicted trends in active site reactivity and to reveal unexpected features of active site architecture.

Hydrogen–Deuterium Exchange of Lipoxygenase Uncovers a Relationship between Distal, Solvent Exposed Protein Motions and the Thermal Activation Barrier for Catalytic Proton-Coupled Electron Tunneling

Defining specific pathways for efficient heat transfer from protein–solvent interfaces to their active sites represents one of the compelling and timely challenges in our quest for a physical description of the origins of enzyme catalysis. Enzymatic hydrogen tunneling reactions constitute excellent systems in which to validate experimental approaches to this important question, given the inherent temperature independence of quantum mechanical wave function overlap. Herein, we present the application of hydrogen–deuterium exchange coupled to mass spectrometry toward the spatial resolution of protein motions that can be related to an enzyme’s catalytic parameters. Employing the proton-coupled electron transfer reaction of soybean lipoxygenase as proof of principle, we first corroborate the impact of active site mutations on increased local flexibility and, second, uncover a solvent-exposed loop, 15–34 Å from the reactive ferric center whose temperature-dependent motions are demonstrated to mirror the enthalpic barrier for catalytic C–H bond cleavage. A network that connects this surface loop to the active site is structurally identified and supported by changes in kinetic parameters that result from site-specific mutations.

Origins of Enzyme Catalysis: Experimental Findings for C–H Activation, New Models, and Their Relevance to Prevailing Theoretical Constructs

The physical basis for enzymatic rate accelerations is a subject of great fundamental interest and of direct relevance to areas that include the de novo design of green catalysts and the pursuit of new drug regimens. Extensive investigations of C-H activating systems have provided considerable insight into the relationship between an enzymes overall structure and the catalytic chemistry at its active site. This Perspective highlights recent experimental data for two members of distinct, yet iconic C-H activation enzyme classes, lipoxygenases and prokaryotic alcohol dehydrogenases. The data necessitate a reformulation of the dominant textbook definition of biological catalysis. A multidimensional model emerges that incorporates a range of protein motions that can be parsed into a combination of global stochastic conformational thermal fluctuations and local donor-acceptor distance sampling. These motions are needed to achieve a high degree of precision with regard to internuclear distances, geometries, and charges within the active site. The available model also suggests a physical framework for understanding the empirical enthalpic barrier in enzyme-catalyzed processes. We conclude by addressing the often conflicting interface between computational and experimental chemists, emphasizing the need for computation to predict experimental results in advance of their measurement.

Synthesis of site-specifically 13C labeled linoleic acids

Soybean lipoxygenase-1 (SLO-1) catalyzes the C-H abstraction from the reactive carbon (C-11) in linoleic acid as the first and rate-determining step in the formation of alkylhydroperoxides. While previous labeling strategies have focused on deuterium labeling to ascertain the primary and secondary kinetic isotope effects for this reaction, there is an emerging interest and need for selectively enriched 13C isotopologues. In this report, we present synthetic strategies for site-specific 13C labeled linoleic acid substrates. We take advantage of a Corey-Fuchs formyl to terminal 13C-labeled alkyne conversion, using 13CBr4 as the labeling source, to reduce the number of steps from a previous fatty acid 13C synthetic labeling approach. The labeled linoleic acid substrates are useful as nuclear tunneling markers and for extracting active site geometries of the enzyme-substrate complex in lipoxygenase.

Redox-Dependent Structural Coupling between the α2 and β2 Subunits in E. coli Ribonucleotide Reductase

Ribonucleotide reductase (RNR) catalyzes the production of deoxyribonucleotides in all cells. In E. coli class Ia RNR, a transient α2β2 complex forms when a ribonucleotide substrate, such as CDP, binds to the α2 subunit. A tyrosyl radical (Y122O•)-diferric cofactor in β2 initiates substrate reduction in α2 via a long-distance, proton-coupled electron transfer (PCET) process. Here, we use reaction-induced FT-IR spectroscopy to describe the α2β2 structural landscapes, which are associated with dATP and hydroxyurea (HU) inhibition. Spectra were acquired after mixing E. coli α2 and β2 with a substrate, CDP, and the allosteric effector, ATP. Isotopic chimeras, (13)Cα2β2 and α2(13)Cβ2, were used to define subunit-specific structural changes. Mixing of α2 and β2 under turnover conditions yielded amide I (C═O) and II (CN/NH) bands, derived from each subunit. The addition of the inhibitor, dATP, resulted in a decreased contribution from amide I bands, attributable to β strands and disordered structures. Significantly, HU-mediated reduction of Y122O• was associated with structural changes in α2, as well as β2. To define the spectral contributions of Y122O•/Y122OH in the quaternary complex, (2)H4 labeling of β2 tyrosines and HU editing were performed. The bands of Y122O•, Y122OH, and D84, a unidentate ligand to the diferric cluster, previously identified in isolated β2, were observed in the α2β2 complex. These spectra also provide evidence for a conformational rearrangement at an additional β2 tyrosine(s), Yx, in the α2β2/CDP/ATP complex. This study illustrates the utility of reaction-induced FT-IR spectroscopy in the study of complex enzymes.

First Site-Specific Incorporation of a Noncanonical Amino Acid into the Photosynthetic Oxygen-Evolving Complex

In photosystem II (PSII), water is oxidized at the oxygen-evolving complex. This process occurs through a light-induced cycle that produces oxygen and protons. While coupled proton and electron transfer reactions play an important role in PSII and other proteins, direct detection of internal proton transfer reactions is challenging. Here, we demonstrate that the unnatural amino acid, 7-azatryptophan (7AW), has unique, pH-sensitive vibrational frequencies, which are sensitive markers of proton transfer. The intrinsically disordered, PSII subunit, PsbO, which contains a single W residue (Trp241), was engineered to contain 7AW at position 241. Fluorescence shows that 7AW-241 is buried in a hydrophobic environment. Reconstitution of 7AW(241)PsbO to PSII had no significant impact on oxygen evolution activity or flash-dependent protein dynamics. We conclude that directed substitution of 7AW into other structural domains is likely to provide a nonperturbative spectroscopic probe, which can be used to define internal proton pathways in PsbO.