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

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

On the occurrence of cytochrome P450 in viruses

Author Posting. © The Author(s), 2019. This is the author's version of the work. It is posted here by permission of National Academy of Sciences for personal use, not for redistribution. The definitive version was published in Proceedings of the National Academy of Sciences of the United States of America 116(25), (2019):12343-12352, doi:10.1073/pnas.1901080116.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.We thank Dr. David Nes (Texas Tech University) for providing sterols and Dr. Matthieu Legendre and Dr. Chantal Abergel (CNRS, Marseille) for access to the P. celtis sequences. Drs. Irina Arkhipova, Mark Hahn, Judith Luborsky, and Ann Bucklin commented on the manuscript. The research was supported by a USA-UK Fulbright Scholarship and a Royal Society grant (to D.C.L.), the Boston University Superfund Research Program [NIH Grant 5P42ES007381 (to J.J.S. and J.V.G.) and NIH Grant 5U41HG003345 (to J.V.G.)], the European Regional Development Fund and Welsh Government Project BEACON (S.L.K.), the Woods Hole Center for Oceans and Human Health [NIH Grant P01ES021923 and National Science Foundation Grant OCE-1314642 (to J.J.S.)], and NIH Grant R01GM53753 (to T.L.P.).2019-12-0

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 timeresolved 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

Cytochrome P450: Nature's Aircraft Carrier

Cytochromes P450 are heme-containing enzymes that utilize O2 for C–H bond activation and play essential roles in drug detoxification and biosynthesis of steroids and a variety of natural products. A number of P450s now have been shown to adopt both an open and closed conformational state. In the open state, the active site is solvent exposed. Upon substrate binding, the P450 shifts to the closed state and sequesters the active site from bulk solvent. Since its discovery, cytochrome P450cam has served as a paradigm for mechanistic and structure-function studies. Over many years of investigation, a wealth of data has suggested that P450cam may possess two camphor binding sites, the active site pocket and an additional site that shifts P450cam toward the open state. However, location of this secondary site was never determined. Here, molecular dynamics simulations were performed that revealed the location of a secondary site on the surface of P450cam. Binding to this allosteric site assists in the opening of both the primary and new secondary active site access channel. Related to these observations is the recent finding that the binding of P450cam’s redox partner, Pdx, favors the open conformation. This shift towards the open state has led to the hypothesis that in order to provide the proton relay network required for O2 activation, P450cam must undergo a structural rearrangement from the closed form. Here, we present the X-ray crystal structure of P450cam complexed with its redox partner, Pdx, substrate, and cyanide as a mimic of a critical intermediate of the catalytic cycle, the “oxy-complex”. The structure of P450cam undergoes ordered changes proposed originally by NMR but never observed crystallographically. These changes provide a channel for water entry and product egress in agreement with the channel formation hypothesized by our simulations. These redox partner interactions studies are extended to a homologue of P450cam, P450terp, which exhibits a less stringent selectivity for its native redox partner Tdx, whose binding may still induce a conformational change in P450terp. Finally, a new P450, CYP102L1, was classified and characterized from Mycobacterium phage Adler and its potential role in viruses is discussed

Effects of Range and Frequency on DIDSON Measurement Accuracy

The DIDSON Sonar is a multi-beam acoustic camera that produces near video quality images in two frequencies (low-1.1MHz and high- 1.8MHz). DIDSON is particularly useful in murky or rough waters because targets within the field-of-view are imaged through sound waves rather than traditional video imagery. Although studies have been conducted as to the accuracy and precision of estimating length with the DIDSON, none in which range and frequency were analyzed simultaneously have been conducted. Our artificial target was manually operated in a controlled pool environment and lengths were measured by the Fish-Marking tool in the DIDSON software. We had four test groups: low frequency at 5 m, high frequency at 5 m, low frequency at 10 m and high frequency at 10 m. The statistical analysis revealed that target range had a significant effect on DIDSON derived measurements while frequency did not. DIDSON-derived length measurements were significantly smaller than the actual target length at both ranges tested

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 Natures 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

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

Effects of Range and Frequency on DIDSON Measurement Accuracy

The DIDSON Sonar is a multi-beam acoustic camera that produces near video quality images in two frequencies (low-1.1MHz and high- 1.8MHz). DIDSON is particularly useful in murky or rough waters because targets within the field-of-view are imaged through sound waves rather than traditional video imagery. Although studies have been conducted as to the accuracy and precision of estimating length with the DIDSON, none in which range and frequency were analyzed simultaneously have been conducted. Our artificial target was manually operated in a controlled pool environment and lengths were measured by the Fish-Marking tool in the DIDSON software. We had four test groups: low frequency at 5 m, high frequency at 5 m, low frequency at 10 m and high frequency at 10 m. The statistical analysis revealed that target range had a significant effect on DIDSON derived measurements while frequency did not. DIDSON-derived length measurements were significantly smaller than the actual target length at both ranges tested