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Open Access Publications from the University of California

Scholarly Works (109 results)

  • Article
  • Peer Reviewed
UC Irvine Previously Published Works (2018)
    Cover page: Structural biology of redox partner binding: Simple and complicatedCreative Commons 'BY' version 4.0 license
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2014)
      Cover page: Heme enzymes: Structure, oxygen activation, and electron transferCreative Commons 'BY' version 4.0 license
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2013)
      Although CYP101D1 and P450cam catalyze the same reaction at similar rates and share strikingly similar active site architectures, there are significant functional differences. CYP101D1 thus provides an opportunity to probe what structural and functional features must be shared and what features can differ but maintain the high catalytic efficiency. Crystal structures of the cyanide complex of wild-type CYP101D1 and it active site mutants, D259N and T260A, have been determined. The conformational changes in CYP101D1 upon cyanide binding are very similar to those of P450cam, indicating a similar mechanism for proton delivery during oxygen activation using solvent-assisted proton transfer. The D259N-CN- complex shows a perturbed solvent structure compared to that of the wild type, which is similar to what was observed in the oxy complex of the corresonding D251N mutant in P450cam. As in P450cam, the T260A mutant is highly uncoupled while the D259N mutant gives barely detectable activity. Despite these similarities, CYP101D1 is able to use the P450cam redox partners while P450cam cannot use the CYP101D1 redox partners. Thus, the strict requirement of P450cam for its own redox partner is relaxed in CYP101D1. Differences in the local environment of the essential Asp (Asp259 in CYP101D1) provide a strucutral basis for understanding these functional differences.
        Cover page: Crystal structures and functional characterization of wild-type CYP101D1 and its active site mutants.
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2015)
        Cytochrome P450cam catalyzes the stereo and regiospecific hydroxylation of camphor to 5-exo-hydroxylcamphor. The two electrons for the oxidation of camphor are provided by putidaredoxin (Pdx), a Fe2 S2 containing protein. Two recent crystal structures of the P450cam-Pdx complex, one solved with the aid of covalent cross-linking and one without, have provided a structural picture of the redox partner interaction. To study the stability of the complex structure and the minor differences between the recent crystal structures, a 100 nanosecond molecular dynamics (MD) simulation of the cross-linked structure, mutated in silico to wild type and the linker molecule removed, was performed. The complex was stable over the course of the simulation though conformational changes including the movement of the C helix of P450cam further toward Pdx allowed for the formation of a number of new contacts at the complex interface that remained stable throughout the simulation. While several minor crystal contacts were lost in the simulation, all major contacts that had been experimentally studied previously were maintained. The equilibrated MD structure contained a mixture of contacts resembling both the cross-linked and noncovalent structures and the newly identified interactions. Finally, the reformation of the P450cam Asp251-Arg186 ion pair in the MD simulation mirrors the ion pair observed in the more promiscuous CYP101D1 and suggests that the Asp251-Arg186 ion pair may be important.
          Cover page: Molecular dynamics of the P450cam-Pdx complex reveals complex stability and novel interface contacts.
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2014)
          The review focuses on those enzymes that catalyze oxidation reactions and those for which crystal structures are available. There are two broad classes of heme enzyme oxidants: oxygenases that use O2 to oxidize, oxygenate, substrates and peroxidases that use 2O2 to oxidize. The review demonstrates that out of the oxidants molecular oxygen is the most unusual, as O2 is not a reactive molecule despite the oxidation of nearly all biological molecules by O2 being a thermodynamically favorable process. The reason is that there is a large kinetic barrier to these reactions owing to O2 being a paramagnetic molecule so that the reaction between a majority of biological molecules that have paired spins is a spin forbidden process.
            Cover page: Heme Enzyme Structure and FunctionCreative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2022)
            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.
              Cover page: Updating the Paradigm: Redox Partner Binding and Conformational Dynamics in Cytochromes P450.
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2018)
              CooAs are dimeric bacterial CO-sensing transcription factors that activate a series of enzymes responsible for CO oxidation. The crystal structure of Rhodospirillum rubrum (rrCooA) shows that the N-terminal Pro from monomer A of the dimer coordinates the heme of monomer B that locks rrCooA in the off state. When CO binds, it is postulated that the Pro is replaced with CO, resulting in a very large reorientation of the DNA binding domains required for specific binding to DNA. Crystal structures of the closely related CooA from Carboxydothermus hydrogenoformans (chCooA) are available, and in one of these, the CO-bound on-state indicates that the N-terminal region that is displaced when CO binds provides contacts between the heme and DNA binding domains that hold the DNA binding domain in position for DNA binding. This has been termed the N-terminal velcro model of CooA activation. The study presented here tests this hypothesis by generating a disulfide mutant that covalently locks chCooA in the on-state. A simple fluorescence assay was used to measure DNA binding, and the S-S mutant was found to be in the on-state even without CO. We also determined the high-resolution crystal structure of the apo-heme domain, and the resulting structure is very similar to the holo-heme-bound structure. This result shows that the heme binding motif forms a stable structure without heme or the DNA binding domain.
                Cover page: Testing the N-Terminal Velcro Model of CooA Carbon Monoxide Activation.
                • Article
                • Peer Reviewed
                UC Irvine Previously Published Works (2018)
                We have compared the thermodynamics of substrate and redox partner binding of P450cam to its close homologue, CYP101D1, using isothermal titration calorimetry (ITC). CYP101D1 binds camphor about 10-fold more weakly than P450cam which is consistent with the inability of camphor to cause a complete low- to high-spin shift in CYP101D1. Even so molecular dynamics simulations show that camphor is very stable in the CYP101D1 active site similar to P450cam. ITC data on the binding of the CYP101D1 ferredoxin redox partner (abbreviated Arx) shows that the substrate-bound closed state of CYP101D1 binds Arx more tightly than the substrate-free open form. This is just the opposite to P450cam where Pdx (ferredoxin redox partner of P450cam) favors binding to the P450cam open state. In addition, CYP101D1-Arx binding has a large negative ΔS while the P450cam-Pdx has a much smaller ΔS indicating that interactions at the docking interface are different. The most obvious difference is that PDXD38 which forms an important ion pair with P450camR112 at the center of the interface is ArxL39 in Arx. This suggests that Arx may adopt a different orientation than Pdx in order to optimize nonpolar interactions with ArxL39.
                  Cover page: Effect of redox partner binding on CYP101D1 conformational dynamics
                  • Thesis
                  • Peer Reviewed
                  UC Irvine Electronic Theses and Dissertations (2016)

                  While ubiquitous in the biological realm, heme enzymes exhibit numerous functions, including protecting organisms against reactive oxygen species, detoxifying xenobiotics and neuronal signaling. The specific function of a heme enzyme is tuned by the structure of the protein that binds the prosthetic group. This relationship between heme and protein is why heme enzymes have long served a foundational role in probing structure-function relationships in biology. The aim of this dissertation is to gain additional insights into heme enzyme structure-function relationships, as well as to gain a better understanding of heme enzyme active site chemistry and the interactions between the enzymes and their redox partners. We have done this by studying four different heme enzyme systems: i) a cytochrome c peroxidase from Leishmania major dubbed Leishmania major Peroxidase (LmP), ii) a speculative peroxynitrite isomerase, also from L. major dubbed pseudoperoxidase (LmPP), iii) a cytochrome P450 monooxygenase from Citrobacter braakii called P450cin, and iv) several mammalian nitric oxide synthase (NOS) isoforms. We determined the precise catalytic mechanism LmP, including the intriguing rate-limiting step of catalysis at steady-state and the importance of proton transfer in the catalytic cycle. We also captured the X-ray crystal structure of the first catalytic intermediate with an iron center unreduced by electrons. This pristine structure, taken together with kinetics, corroborates our mechanistic conclusions. In the process, we validated the usefulness of X-ray free electron lasers as a powerful tool to probe the structure of enzymes that contain transition metals. We probed the association of LmP with its redox partner LmCytc and discovered the existence of a non-catalytic binding site. For our second system, we have solved the crystal structure of a novel LmPP and proposed a hypothetical mechanism that awaits testing. Although we were unable to reach our goals for our third system, we established a protocol to address the crystallization of the P450cin-Cdx complex. For our fourth system, we explored the precise and sensitive balance of forces that stabilize the structure and thus function of NOS, which is maintained by an elegant synergistic relationship between the Zn2+, cofactor, and substrate binding sites. We finish by describing collaborative work attempting to design potent, isomer selective and bioavailable inhibitors to treat neurodegenerative diseases.

                    Cover page: Exploring Structure-Function Relationships and Redox Partner Interactions in Heme EnzymesCreative Commons 'BY-NC-ND' version 4.0 license
                    • Thesis
                    • Peer Reviewed
                    UC Irvine Electronic Theses and Dissertations (2020)

                    Antibiotic resistance of pathogenic bacteria is a growing problem of global concern. Current antibiotics target all bacteria indiscriminately and consequently wipe out good, beneficial bacteria as well as the bad, pathogenic ones. Bacterial Nitric Oxide Synthase (bNOS) has been identified as a suitable target to combat Methicilin Resistant Staphylococcus aureus (MRSA), a pathogen responsible for thousands of deaths each year in the United States alone. We report here the development of bNOS selective inhibitors based on an aminoquinolone chemical scaffold. These inhibitors were found to bind in both the active and pterin sites of bNOS, effectively reducing bacterial survival. In addition, we tested the limits of an NO selective chemical probe and began preliminary investigations of other potential drug targets for MRSA related to the NO pathway.

                      Cover page: Bacterial Nitric Oxide Synthase Inhibition: Designer Drugs to Fight Bad BugsCreative Commons 'BY-NC-ND' version 4.0 license
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