Changes in an enzyme ensemble during catalysis observed by high-resolution XFEL crystallography.

Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

Changes in an Enzyme Ensemble During Catalysis Observed by High Resolution XFEL Crystallography.

Enzymes populate ensembles of structures with intrinsically different catalytic proficiencies that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL) to observe catalysis in a designed mutant (G150T) isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations and formation of the thioimidate catalytic intermediate selects for catalytically competent substates. A prior proposal for active site cysteine charge-coupled conformational changes in ICH is validated by determining structures of the enzyme over a range of pH values. A combination of large molecular dynamics simulations of the enzyme in crystallo and time-resolved electron density maps shows that ionization of the general acid Asp17 during catalysis causes additional conformational changes that propagate across the dimer interface, connecting the two active sites. These ionization-linked changes in the ICH conformational ensemble permit water to enter the active site in a location that is poised for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

The role of basicity in selective C-H bond activation by transition metal-oxidos.

The development of (bio)catalysts capable of selectively activating strong C-H bonds is a continuing challenge in modern chemistry. In both metalloenzymes and synthetic systems capable of activating C-H bonds, transition metal-oxido intermediates serve as the active species for reactivity whose thermodynamic properties influence the bond strengths they are capable of activating. In this Frontier article, we present current ideas of how the basicity of transition metal-oxidos impacts their reactivity with C-H bonds and present new opportunities within this field. We highlight recent insights into the role basicity plays in the activation process and its influence on mechanism, as well as the important role that secondary coordination sphere effects, such as hydrogen bonds, in tuning the basicity of the metal-oxido species is discussed.

Selective C-H Bond Cleavage with a High-Spin FeIV-Oxido Complex.

Non-heme Fe monooxygenases activate C-H bonds using intermediates with high-spin FeIV-oxido centers. To mimic these sites, a new tripodal ligand [pop]3- was prepared that contains three phosphoryl amido groups that are capable of stabilizing metal centers in high oxidation states. The ligand was used to generate [FeIVpop(O)]-, a new FeIV-oxido complex with an S = 2 spin ground state. Spectroscopic measurements, which included low-temperature absorption and electron paramagnetic resonance spectroscopy, supported the assignment of a high-spin FeIV center. The complex showed reactivity with benzyl alcohol as the external substrate but not with related compounds (e.g., ethyl benzene and benzyl methyl ether), suggesting the possibility that hydrogen bonding interaction(s) between the substrate and [FeIVpop(O)]- was necessary for reactivity. These results exemplify the potential role of the secondary coordination sphere in metal-mediated processes.

Redox partner recognition and selectivity of cytochrome P450lin (CYP111A1).

The strict requirement of cytochrome P450cam for its native ferredoxin redox partner, putidaredoxin (Pdx), is not exhibited by any other known cytochrome P450 (CYP) system and the molecular details of redox partner selectivity are still not completely understood. We therefore examined the selectivity of a related Pseudomonas cytochrome P450, P450lin, by testing its activity with non-native redox partners. We found that P450lin could utilize Arx, the native redox partner of CYP101D1, to enable turnover of its substrate, linalool, while Pdx showed limited activity. Arx exhibited a higher sequence similarity to P450lins native redox partner, linredoxin (Ldx) than Pdx, including several residues that are believed to be at the interface of the two proteins, based on the P450cam-Pdx complex structure. We therefore mutated Pdx to resemble Ldx and Arx and found that a double mutant, D38L/∆106, displayed higher activity than Arx. In addition, Pdx D38L/∆106 does not induce a low-spin shift in linalool bound P450lin but does destabilize the P450lin-oxycomplex. Together our results suggest that P450lin and its redox partners may form a similar interface to P450cam-Pdx, but the interactions that allow for productive turnover are different.

Cooperative Substrate Binding Controls Catalysis in Bacterial Cytochrome P450terp (CYP108A1).

Despite being one of the most well-studied aspects of cytochrome P450 chemistry, important questions remain regarding the nature and ubiquity of allosteric regulation of catalysis. The crystal structure of a bacterial P450, P450terp, in the presence of substrate reveals two binding sites, one above the heme in position for regioselective hydroxylation and another in the substrate access channel. Unlike many bacterial P450s, P450terp does not exhibit an open to closed conformational change when substrate binds; instead, P450terp uses the second substrate molecule to hold the first substrate molecule in position for catalysis. Spectral titrations clearly show that substrate binding to P450terp is cooperative with a Hill coefficient of 1.4 and is supported by isothermal titration calorimetry. The importance of the allosteric site was explored by a series of mutations that weaken the second site and that help hold the first substrate in position for proper catalysis. We further measured the coupling efficiency of both the wild-type (WT) enzyme and the mutant enzymes. While the WT enzyme exhibits 97% efficiency, each of the variants showed lower catalytic efficiency. Additionally, the variants show decreased spin shifts upon binding of substrate. These results are the first clear example of positive homotropic allostery in a class 1 bacterial P450 with its natural substrate. Combined with our recent results from P450cam showing complex substrate allostery and conformational dynamics, our present study with P450terp indicates that bacterial P450s may not be as simple as once thought and share complex substrate binding properties usually associated with only mammalian P450s.

µ-Oxo Dimerization Effects on Ground- and Excited-State Properties of a Water-Soluble Iron Porphyrin CO2 Reduction Catalyst.

Iron 5,10,15,20-tetra(para-N,N,N-trimethylanilinium)porphyrin (Fe-p-TMA) is a water-soluble catalyst capable of electrochemical and photochemical CO2 reduction. Although its catalytic ability has been thoroughly investigated, the mechanism and associated intermediates are largely unknown. Previous studies proposed that Fe-p-TMA enters catalytic cycles as a monomeric species. However, we demonstrate herein that, in aqueous solutions, Fe-p-TMA undergoes formation of a µ-oxo porphyrin dimer that exists in equilibrium with its monomeric form. The propensity for µ-oxo formation is highly dependent on the solution pH and ionic strength. Indeed, the µ-oxo form is stabilized in the presence of electrolytes that are key components of catalytically relevant conditions. By leveraging the ability to chemically control and spectrally address both species, we characterize their ground-state electronic structures and excited-state photodynamics. Global fitting of ultrafast transient absorption data reveals two distinct excited-state relaxation pathways: a three-component sequential model consistent with monomeric relaxation and a two-component sequential model for the µ-oxo species. Relaxation of the monomeric species is best described as a ligand-to-metal charge transfer (τ1 = ∼500 fs), an ionic strength-dependent metal-to-ligand charge transfer (τ2 = 2-4 ps), and finally relaxation of a ligand field excited state to the ground state (τ3 = 5 ps). Conversely, excited-state relaxation of the µ-oxo species proceeds via cleavage of an FeIII-O bond to generate transient FeIV═O and FeII porphyrin species (τ1 = 2 ps) that recombine to the ground-state µ-oxo species (τ2 = ∼1 ns). This latter lifetime extends to timescales relevant for chemical reactivity. It is therefore emphasized that further consideration of catalyst speciation and chemical microenvironments is necessary for elucidating the mechanisms of catalytic CO2 reduction reactions.

Updating the Paradigm: Redox Partner Binding and Conformational Dynamics in Cytochromes P450.

This Account summarizes recent findings centered on the role that redox partner binding, allostery, and conformational dynamics plays in cytochrome P450 proton coupled electron transfer. P450s are one of Nature's largest enzyme families and it is not uncommon to find a P450 wherever substrate oxidation is required in the formation of essential molecules critical to the life of the organism or in xenobiotic detoxification. P450s can operate on a remarkably large range of substrates from the very small to the very large, yet the overall P450 three-dimensional structure is conserved. Given this conservation of structure, it is generally assumed that the basic catalytic mechanism is conserved. In nearly all P450s, the O2 O-O bond must be cleaved heterolytically enabling one oxygen atom, the distal oxygen, to depart as water and leave behind a heme iron-linked O atom as the powerful oxidant that is used to activate the nearby substrate. For this process to proceed efficiently, externally supplied electrons and protons are required. Two protons must be added to the departing O atom while an electron is transferred from a redox partner that typically contains either a Fe2S2 or FMN redox center. The paradigm P450 used to unravel the details of these mechanisms has been the bacterial CYP101A1 or P450cam. P450cam is specific for its own Fe2S2 redox partner, putidaredoxin or Pdx, and it has long been postulated that Pdx plays an effector/allosteric role by possibly switching P450cam to an active conformation. Crystal structures, spectroscopic data, and direct binding experiments of the P450cam-Pdx complex provide some answers. Pdx shifts the conformation of P450cam to a more open state, a transition that is postulated to trigger the proton relay network required for O2 activation. An essential part of this proton relay network is a highly conserved Asp (sometimes Glu) that is known to be critical for activity in a number of P450s. How this Asp and proton delivery networks are connected to redox partner binding is quite simple. In the closed state, this Asp is tied down by salt bridges, but these salt bridges are ruptured when Pdx binds, leaving the Asp free to serve its role in proton transfer. An alternative hypothesis suggests that a specific proton relay network is not really necessary. In this scenario, the Asp plays a structural role in the open/close transition and merely opening the active site access channel is sufficient to enable solvent protons in for O2 protonation. Experiments designed to test these various hypotheses have revealed some surprises in both P450cam and other bacterial P450s. Molecular dynamics and crystallography show that P450cam can undergo rather significant conformational gymnastics that result in a large restructuring of the active site requiring multiple cis/trans proline isomerizations. It also has been found that X-ray driven substrate hydroxylation is a useful tool for better understanding the role that the essential Asp and surrounding residues play in catalysis. Here we summarize these recent results which provide a much more dynamic picture of P450 catalysis.

Partial Opening of Cytochrome P450cam (CYP101A1) Is Driven by Allostery and Putidaredoxin Binding.

Cytochrome P450cam (CYP101A1) catalyzes the regio- and stereo-specific 5-exo-hydroxylation of camphor via a multistep catalytic cycle that involves two-electron transfer steps, with an absolute requirement that the second electron be donated by the ferrodoxin, putidaredoxin (Pdx). Whether P450cam, once camphor has bound to the active site and the substrate entry channel has closed, opens up upon Pdx binding, during the second electron transfer step, or it remains closed is still a matter of debate. A potential allosteric site for camphor binding has been identified and postulated to play a role in the binding of Pdx. Here, we have revisited paramagnetic NMR spectroscopy data and determined a heterogeneous ensemble of structures that explains the data, provides a complete representation of the P450cam/Pdx complex in solution, and reconciles alternative hypotheses. The allosteric camphor binding site is always present, and the conformational changes induced by camphor binding to this site facilitates Pdx binding. We also determined that the state to which Pdx binds comprises an ensemble of structures that have features of both the open and closed state. These results demonstrate that there is a finely balanced interaction between allosteric camphor binding and the binding of Pdx at high camphor concentrations.

Artificial Metalloproteins with Dinuclear Iron-Hydroxido Centers.

Dinuclear iron centers with a bridging hydroxido or oxido ligand form active sites within a variety of metalloproteins. A key feature of these sites is the ability of the protein to control the structures around the Fe centers, which leads to entatic states that are essential for function. To simulate this controlled environment, artificial proteins have been engineered using biotin-streptavidin (Sav) technology in which Fe complexes from adjacent subunits can assemble to form [FeIII-(µ-OH)-FeIII] cores. The assembly process is promoted by the site-specific localization of the Fe complexes within a subunit through the designed mutation of a tyrosinate side chain to coordinate the Fe centers. An important outcome is that the Sav host can regulate the Fe···Fe separation, which is known to be important for function in natural metalloproteins. Spectroscopic and structural studies from X-ray diffraction methods revealed uncommonly long Fe···Fe separations that change by less than 0.3 Å upon the binding of additional bridging ligands. The structural constraints imposed by the protein host on the di-Fe cores are unique and create examples of active sites having entatic states within engineered artificial metalloproteins.

Understanding Covalent versus Spin-Orbit Coupling Contributions to Temperature-Dependent Electron Spin Relaxation in Cupric and Vanadyl Phthalocyanines.

Recent interest in transition-metal complexes as potential quantum bits (qubits) has reinvigorated the investigation of fundamental contributions to electron spin relaxation in various ligand scaffolds. From quantum computers to chemical and biological sensors, interest in leveraging the quantum properties of these molecules has opened a discussion of the requirements to maintain coherence over a large temperature range, including near room temperature. Here we compare temperature-, magnetic field position-, and concentration-dependent electron spin relaxation in copper(II) phthalocyanine (CuPc) and vanadyl phthalocyanine (VOPc) doped into diamagnetic hosts. While VOPc demonstrates coherence up to room temperature, CuPc coherence times become rapidly T1-limited with increasing temperature, despite featuring a more covalent ground-state wave function than VOPc. As rationalized by a ligand field model, this difference is ascribed to different spin-orbit coupling (SOC) constants for Cu(II) versus V(IV). The manifestation of SOC contributions to spin-phonon coupling and electron spin relaxation in different ligand fields is discussed, allowing for a further understanding of the competing roles of SOC and covalency in electron spin relaxation.

Artificial Iron Proteins: Modeling the Active Sites in Non-Heme Dioxygenases.

An important class of non-heme dioxygenases contains a conserved Fe binding site that consists of a 2-His-1-carboxylate facial triad. Results from structural biology show that, in the resting state, these proteins are six-coordinate with aqua ligands occupying the remaining three coordination sites. We have utilized biotin-streptavidin (Sav) technology to design new artificial Fe proteins (ArMs) that have many of the same structural features found within active sites of these non-heme dioxygenases. An Sav variant was isolated that contains the S112E mutation, which installed a carboxylate side chain in the appropriate position to bind to a synthetic FeII complex confined within Sav. Structural studies using X-ray diffraction (XRD) methods revealed a facial triad binding site that is composed of two N donors from the biotinylated ligand and the monodentate coordination of the carboxylate from S112E. Two aqua ligands complete the primary coordination sphere of the FeII center with both involved in hydrogen bond networks within Sav. The corresponding FeIII protein was also prepared and structurally characterized to show a six-coordinate complex with two exogenous acetato ligands. The FeIII protein was further shown to bind an exogenous azido ligand through replacement of one acetato ligand. Spectroscopic studies of the ArMs in solution support the results found by XRD.

On the occurrence of cytochrome P450 in viruses.

Genes encoding cytochrome P450 (CYP; P450) enzymes occur widely in the Archaea, Bacteria, and Eukarya, where they play important roles in metabolism of endogenous regulatory molecules and exogenous chemicals. We now report that genes for multiple and unique P450s occur commonly in giant viruses in the Mimiviridae, Pandoraviridae, and other families in the proposed order Megavirales. P450 genes were also identified in a herpesvirus (Ranid herpesvirus 3) and a phage (Mycobacterium phage Adler). The Adler phage P450 was classified as CYP102L1, and the crystal structure of the open form was solved at 2.5 Å. Genes encoding known redox partners for P450s (cytochrome P450 reductase, ferredoxin and ferredoxin reductase, and flavodoxin and flavodoxin reductase) were not found in any viral genome so far described, implying that host redox partners may drive viral P450 activities. Giant virus P450 proteins share no more than 25% identity with the P450 gene products we identified in Acanthamoeba castellanii, an amoeba host for many giant viruses. Thus, the origin of the unique P450 genes in giant viruses remains unknown. If giant virus P450 genes were acquired from a host, we suggest it could have been from an as yet unknown and possibly ancient host. These studies expand the horizon in the evolution and diversity of the enormously important P450 superfamily. Determining the origin and function of P450s in giant viruses may help to discern the origin of the giant viruses themselves.

Ligand and Redox Partner Binding Generates a New Conformational State in Cytochrome P450cam (CYP101A1).

It has become increasingly clear that cytochromes P450 can cycle back and forth between two extreme conformational states termed the closed and open states. In the well-studied cytochrome P450cam, the binding of its redox partner, putidaredoxin (Pdx), shifts P450cam toward the open state. Shifting to the open state is thought to be important in the formation of a proton relay network essential for O-O bond cleavage and formation of the active Fe(IV)═O intermediate. Another important intermediate is the oxy-P450cam complex when bound to Pdx. Trapping this intermediate in crystallo is challenging owing to its instability, but the CN- complex is both stable and an excellent mimic of the O2 complex. Here we present the P450cam-Pdx structure complexed with CN-. CN- results in large conformational changes including cis/trans isomerization of proline residues. Changes include large rearrangements of active-site residues and the formation of new active-site access channel that we have termed channel 2. The formation of channel 2 has also been observed in our previous molecular dynamics simulations wherein substrate binding to an allosteric site remote from the active site opens up channel 2. This new structure supports an extensive amount of previous work showing that distant regions of the structure are dynamically coupled and underscores the potentially important role that large conformational changes and dynamics play in P450 catalysis.

Substrate-Dependent Allosteric Regulation in Cytochrome P450cam (CYP101A1).

Various biophysical methods have provided evidence of a second substrate binding site in the well-studied cytochrome P450cam, although the location and biological relevance of this site has remained elusive. A related question is how substrate and product binding and egress occurs. While many active site access channels have been hypothesized, only one, channel 1, has been experimentally validated. In this study, molecular dynamics simulations reveal an allosteric site related to substrate binding and product egress. The remote allosteric site opens channel 1 and primes the formation of a new channel that is roughly perpendicular to channel 1. Substrate entry to the active site via channel 1 as well as substrate/product egress via channel 2 is observed after binding of a second molecule of substrate to the allosteric site, indicating cooperativity between these two sites. These results are consistent with and bring together many early and recent experimental results to reveal a dynamic interplay between a weak allosteric site and substrate binding to the active site that controls P450cam activity.