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.

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 O 2 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.

Membrane proteins, due to their roles as cell receptors and signaling mediators, make prime candidates for drug targets. The computational analysis of protein-ligand binding affinities has been widely employed as a tool in rational drug design efforts. Although efficient implicit solvent-based methods for modeling globular protein-ligand binding have been around for many years, the extension of such methods to membrane protein-ligand binding is still in its infancy. In this study, we extended the widely used Amber/MMPBSA method to model membrane protein-ligand systems, and we used it to analyze protein-ligand binding for the human purinergic platelet receptor (P2Y12R), a prominent drug target in the inhibition of platelet aggregation for the prevention of myocardial infarction and stroke. The binding affinities, computed by the Amber/MMPBSA method using standard parameters, correlate well with experiment. A detailed investigation of these parameters was conducted to assess their impact on the accuracy of the method. These analyses show the importance of properly treating the non-polar solvation interactions and the electrostatic polarization in the binding of nucleotide agonists and non-nucleotide antagonists to P2Y12R. Based on the crystal structures and the experimental conditions in the binding assay, we further hypothesized that the nucleotide agonists lose their bound magnesium ion upon binding to P2Y12R, and our computational study supports this hypothesis. Ultimately, this work illustrates the value of computational analysis in the interpretation of experimental binding reactions.

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 T 1-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.

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.