Low-temperature trapping of N2 reduction reaction intermediates in nitrogenase MoFe protein-CdS quantum dot complexes.

The biological reduction of N2 to ammonia requires the ATP-dependent, sequential delivery of electrons from the Fe protein to the MoFe protein of nitrogenase. It has been demonstrated that CdS nanocrystals can replace the Fe protein to deliver photoexcited electrons to the MoFe protein. Herein, light-activated electron delivery within the CdS:MoFe protein complex was achieved in the frozen state, revealing that all the electron paramagnetic resonance (EPR) active E-state intermediates in the catalytic cycle can be trapped and characterized by EPR spectroscopy. Prior to illumination, the CdS:MoFe protein complex EPR spectrum was composed of a S = 3/2 rhombic signal (g = 4.33, 3.63, and 2.01) consistent with the FeMo-cofactor in the resting state, E0. Illumination for sequential 1-h periods at 233 K under 1 atm of N2 led to a cumulative attenuation of E0 by 75%. This coincided with the appearance of S = 3/2 and S = 1/2 signals assigned to two-electron (E2) and four-electron (E4) reduced states of the FeMo-cofactor, together with additional S = 1/2 signals consistent with the formation of E6 and E8 states. Simulations of EPR spectra allowed quantification of the different E-state populations, along with mapping of these populations onto the Lowe-Thorneley kinetic scheme. The outcome of this work demonstrates that the photochemical delivery of electrons to the MoFe protein can be used to populate all of the EPR active E-state intermediates of the nitrogenase MoFe protein cycle.

Cryo-annealing of Photoreduced CdS Quantum Dot-Nitrogenase MoFe Protein Complexes Reveals the Kinetic Stability of the E4(2N2H) Intermediate.

A critical step in the mechanism of N2 reduction to 2NH3 catalyzed by the enzyme nitrogenase is the reaction of the four-electron/four-proton reduced intermediate state of the active-site FeMo-cofactor (E4(4H)). This state is a junction in the catalytic mechanism, either relaxing by the reaction of a metal bound Fe-hydride with a proton forming H2 or going forward with N2 binding coupled to the reductive elimination (re) of two Fe-hydrides as H2 to form the E4(2N2H) state. E4(2N2H) can relax to E4(4H) by the oxidative addition (oa) of H2 and release of N2 or can be further reduced in a series of catalytic steps to release 2NH3. If the H2 re/oa mechanism is correct, it requires that oa of H2 be associative with E4(2N2H). In this report, we have taken advantage of CdS quantum dots in complex with MoFe protein to achieve photodriven electron delivery in the frozen state, with cryo-annealing in the dark, to reveal details of the E-state species and to test the stability of E4(2N2H). Illumination of frozen CdS:MoFe protein complexes led to formation of a population of reduced intermediates. Electron paramagnetic resonance spectroscopy identified E-state signals including E2 and E4(2N2H), as well as signals suggesting the formation of E6 or E8. It is shown that in the frozen state when pN2 is much greater than pH2, the E4(2N2H) state is kinetically stable, with very limited forward or reverse reaction rates. These results establish that the oa of H2 to the E4(2N2H) state follows an associative reaction mechanism.

Dissecting Electronic-Structural Transitions in the Nitrogenase MoFe Protein P-Cluster during Reduction.

The [8Fe-7S] P-cluster of nitrogenase MoFe protein mediates electron transfer from nitrogenase Fe protein during the catalytic production of ammonia. The P-cluster transitions between three oxidation states, PN, P+, P2+ of which PN↔P+ is critical to electron exchange in the nitrogenase complex during turnover. To dissect the steps in formation of P+ during electron transfer, photochemical reduction of MoFe protein at 231-263 K was used to trap formation of P+ intermediates for analysis by EPR. In complexes with CdS nanocrystals, illumination of MoFe protein led to reduction of the P-cluster P2+ that was coincident with formation of three distinct EPR signals: S = 1/2 axial and rhombic signals, and a high-spin S = 7/2 signal. Under dark annealing the axial and high-spin signal intensities declined, which coincided with an increase in the rhombic signal intensity. A fit of the time-dependent changes of the axial and high-spin signals to a reaction model demonstrates they are intermediates in the formation of the P-cluster P+ resting state and defines how spin-state transitions are coupled to changes in P-cluster oxidation state in MoFe protein during electron transfer.

Surface-Ligand "Liquid" to "Crystalline" Phase Transition Modulates the Solar H2 Production Quantum Efficiency of CdS Nanorod/Mediator/Hydrogenase Assemblies.

This study reports how the length of capping ligands on a nanocrystal surface affects its interfacial electron transfer (ET) with surrounding molecular electron acceptors, and consequently, impact the H2 production of a biotic-abiotic hybrid artificial photosynthetic system. Specifically, we study how the H2 production efficiency of a hybrid system, combining CdS nanorods (NRs), [NiFe] hydrogenase, and redox mediators (propyl-bridged 2,2'-bipyridinium, PDQ2+), depends on the alkyl chain length of mercaptocarboxylate ligands on the NR surface. We observe a minor decrease of the quantum yield for H2 production from 54 ± 6 to 43 ± 2% when varying the number of methylene units in the ligands from 2 to 7. In contrast, an abrupt decrease of the yield was observed from 43 ± 2 to 4 ± 1% when further increasing n from 7 to 11. ET studies reveal that the intrinsic ET rates from the NRs to the electron acceptor PDQ2+ are all within 108-109 s-1 regardless of the length of the capping ligands. However, the number of adsorbed PDQ2+ molecules on NR surfaces decreases dramatically when n ≥ 10, with the saturating number changing from 45 ± 5 to 0.3 ± 0.1 for n = 2 and 11, respectively. These results are not consistent with the commonly perceived exponential dependence of ET rates on the ligand length. Instead, they can be explained by the change of the accessibility of NR surfaces to electron acceptors from a disordered "liquid" phase at n < 7 to a more ordered "crystalline" phases at n > ∼7. These results highlight that the order of capping ligands is an important design parameter for further constructing nanocrystal/molecular assemblies in broad nanocrystal-based applications.

Metal-ligand cooperativity in the soluble hydrogenase-1 from Pyrococcus furiosus.

Metal-ligand cooperativity is an essential feature of bioinorganic catalysis. The design principles of such cooperativity in metalloenzymes are underexplored, but are critical to understand for developing efficient catalysts designed with earth abundant metals for small molecule activation. The simple substrate requirements of reversible proton reduction by the [NiFe]-hydrogenases make them a model bioinorganic system. A highly conserved arginine residue (R355) directly above the exogenous ligand binding position of the [NiFe]-catalytic core is known to be essential for optimal function because mutation to a lysine results in lower catalytic rates. To expand on our studies of soluble hydrogenase-1 from Pyrococcus furiosus (Pf SH1), we investigated the role of R355 by site-directed-mutagenesis to a lysine (R355K) using infrared and electron paramagnetic resonance spectroscopic probes sensitive to active site redox and protonation events. It was found the mutation resulted in an altered ligand binding environment at the [NiFe] centre. A key observation was destabilization of the Nia 3+-C state, which contains a bridging hydride. Instead, the tautomeric Nia +-L states were observed. Overall, the results provided insight into complex metal-ligand cooperativity between the active site and protein scaffold that modulates the bridging hydride stability and the proton inventory, which should prove valuable to design principles for efficient bioinspired catalysts.

Localized Nanoscale Heating Leads to Ultrafast Hydrogel Volume-Phase Transition.

The rate of the volume-phase transition for stimuli-responsive hydrogel particles ranging in size from millimeters to nanometers is limited by the rate of water transport, which is proportional to the surface area of the particle. Here, we hypothesized that the rate of volume-phase transition could be accelerated if the stimulus is geometrically controlled from the inside out, thus facilitating outward water ejection. To test this concept, we applied transient absorption spectroscopy, laser temperature-jump spectroscopy, and finite-element analysis modeling to characterize the dynamics of the volume-phase transition of hydrogel particles with a gold nanorod core. Our results demonstrate that the nanoscale heating of the hydrogel particle core led to an ultrafast, 60 ns particle collapse, which is 2-3 orders of magnitude faster than the response generated from conventional heating. This is the fastest recorded response time of a hydrogel material, thus opening potential applications for such stimuli-responsive materials.

Structural Insights into Oxygen-Dependent Signal Transduction within Globin Coupled Sensors.

In order to respond to external stimuli, bacteria have evolved sensor proteins linking external signals to intracellular outputs that can then regulate downstream pathways and phenotypes. Globin coupled sensor proteins (GCSs) serve to link environmental O2 levels to cellular processes by coupling a heme-containing sensor globin domain to a catalytic output domain. However, the mechanism by which O2 binding activates these proteins is currently unknown. To provide insights into the signaling mechanism, two distinct dimeric complexes of the isolated globin domain of the GCS from Bordetella pertussis ( BpeGlobin) were solved via X-ray crystallography in which differences in ligand-bound states were observed. Both monomers of one dimer contain Fe(II)-O2 states, while the other dimer consists of the Fe(III)-H2O and Fe(II)-O2 states. These data provide the first molecular insights into the heme pocket conformation of the active Fe(II)-O2 form of these enzymes. In addition, heme distortion modes and heme-protein interactions were found to correlate with the ligation state of the globin, suggesting that these conformational changes play a role in O2-dependent signaling. Fourier transform infrared spectroscopy (FTIR) of the full-length GCS from B. pertussis ( BpeGReg) and the closely related GCS from Pectobacterium carotovorum ssp. carotovorum ( PccGCS) confirmed the importance of an ordered water within the heme pocket and two distal residues (Tyr43 and Ser68) as hydrogen-bond donors. Taken together, this work provides mechanistic insights into BpeGReg O2 sensing and the signaling mechanisms of diguanylate cyclase-containing GCS proteins.

Applications of Photogating and Time Resolved Spectroscopy to Mechanistic Studies of Hydrogenases.

Rapid and facile redox chemistry is exemplified in nature by the oxidoreductases, the class of enzymes that catalyze electron transfer (ET) from a donor to an acceptor. The key role of oxidoreductases in metabolism and biosynthesis has imposed evolutionary pressure to enhance enzyme efficiency, pushing some toward the diffusion limit. Understanding the detailed molecular mechanisms of these highly optimized enzymes would provide an important foundation for the rational design of catalysts for multielectron chemistry, including fuel production. The hydrogenases (H2ases) are the oxidoreductases that catalyze the most basic electron and proton transfer reactions relevant to fuel production, the interconversion of protons and hydrogen, with kcat > 103 s-1. Thus, they provide a model system for studying the efficiency exhibited by oxidoreductases. Because of the extraordinarily fast catalytic rates of these enzymes, their mechanisms have been difficult to study directly but instead have been inferred from structural and steady-state measurements. Although informative, the kinetic competency of observed equilibrium steps can only be suggested by these methods, not demonstrated, because the fundamental (fast) catalytic steps remain unresolved, resulting in minimal insight regarding the underlying ET and proton transfer (PT) events. Motivated by this gap in understanding, we developed an approach capable of observing elementary ET and PT during such fast enzyme turnover by combining a laser-induced potential jump with time-resolved spectroscopy. The potential jump initiates enzyme turnover by utilizing a short-pulsed laser to release a "caged" electron from a nanomaterial or NAD(P)H, which is then captured by a mediator such as methyl viologen. The subsequent enzyme reduction and turnover are monitored by transient absorption spectroscopy in the visible or mid-IR spectral regions. The method is completely general and in principle can be applied to any catalytic redox reaction. In the case of hydrogenases, time-resolved infrared spectroscopy of the active site CO ligands is particularly informative since the IR frequencies are exquisitely sensitive to the redox and protonation states. Using this methodology, we have developed a description of the catalytic mechanism of the Pyrococcus furiosus [NiFe]-hydrogenase by demonstrating the kinetic and chemical competency of equilibrium states and by invoking new intermediates. Additionally, the pre-steady-state kinetics revealed a distinct role of proton tunneling in concerted electron-proton transfer (EPT) modulated by a conserved glutamic acid residue. Similar multisite EPT processes have been implicated in numerous enzymes but have not been demonstrated explicitly. These methods have also been successfully applied to an electron bifurcating [FeFe]-H2ase from Thermotoga maritima, establishing the kinetic competency of the Hox, Hred, and Hsred intermediates of the [FeFe] enzyme. These results provide fundamental insight on the factors that control low barrier proton and electron flow in enzymes and thus provide a foundation for the rational design of reversible biomimetic catalysts.

Glutamate Gated Proton-Coupled Electron Transfer Activity of a [NiFe]-Hydrogenase.

[NiFe] hydrogenases are metalloenzymes that catalyze the reversible oxidation of H2. While electron transfer to and from the active site is understood to occur through iron-sulfur clusters, the mechanism of proton transfer is still debated. Two mechanisms for proton exchange with the active site have been proposed that involve distinct and conserved ionizable amino acid residues, one a glutamate, and the other an arginine. To examine the potential role of the conserved glutamate on active site acid-base chemistry, we mutated the putative proton donor E17 to Q in the soluble hydrogenase I from Pyrococcus furiosus using site directed mutagenesis. FTIR spectroscopy, sensitive to the CO and CN ligands of the active site, reveals catalytically active species generated upon reduction with H2, including absorption features consistent with the Nia-C intermediate. Time-resolved IR spectroscopy, which probes active site dynamics after hydride photolysis from Nia-C, indicates the E17Q mutation does not interfere with the hydride photolysis process generating known intermediates Nia-I1 and Nia-I2. Strikingly, the E17Q mutation disrupts obligatory proton-coupled electron transfer from the Nia-I1 state, thereby preventing formation of Nia-S. These results directly establish E17 as a proton donor/acceptor in the Nia-S ↔ Nia-C equilibrium.

Globin domain interactions control heme pocket conformation and oligomerization of globin coupled sensors.

Globin coupled sensors (GCS) are O2-sensing proteins used by bacteria to monitor the surrounding gaseous environment. To investigate the biphasic O2 dissociation kinetics observed for full-length GCS proteins, isolated globin domains from Pectobacterium carotovorum ssp. carotovorum (PccGlobin), and Bordetella pertussis (BpeGlobin), have been characterized. PccGlobin is found to be dimeric, while BpeGlobin is monomeric, indicating key differences in the globin domain dimer interface. Through characterization of wild type globin domains and globin variants with mutations at the dimer interface and within the distal pocket, dimerization of the globin domain is demonstrated to correlate with biphasic dissociation kinetics. Furthermore, a distal pocket tyrosine is identified as the primary hydrogen bond donor, while a secondary hydrogen bond donor within the distal heme pocket is involved in conformation(s) that lead to the second O2 dissociation rate. These findings highlight the role of the globin dimer interface in controlling properties of both the heme pocket and full-length GCS proteins.

Proton Inventory and Dynamics in the Nia-S to Nia-C Transition of a [NiFe] Hydrogenase.

Hydrogenases (H2ases) represent one of the most striking examples of biological proton-coupled electron transfer (PCET) chemistry, functioning in facile proton reduction and H2 oxidation involving long-range proton and electron transport. Spectroscopic and electrochemical studies of the [NiFe] H2ases have identified several catalytic intermediates, but the details of their interconversion are still a matter of debate. Here we use steady state and time-resolved infrared spectroscopy, sensitive to the CO ligand of the active site iron, as a probe of the proton inventory as well as electron and proton transfer dynamics in the soluble hydrogenase I from Pyrococcus furiosus. Subtle shifts in infrared signatures associated with the Nia-C and Nia-S states as a function of pH revealed an acid-base equilibrium associated with an ionizable amino acid near the active site. Protonation of this residue was found to correlate with the photoproduct distribution that results from hydride photolysis of the Nia-C state, in which one of the two photoproduct states becomes inaccessible at low pH. Additionally, the ability to generate Nia-S via PCET from Nia-C was weakened at low pH, suggesting prior protonation of the proton acceptor. Kinetic and thermodynamic analysis of electron and proton transfer with respect to the various proton inventories was utilized to develop a chemical model for reversible hydride oxidation involving two intermediates differing in their hydrogen bonding character.