Electronic Structure of Cytochrome P450

The optical properties of P450 have been investigated by means of polarized absorption spectroscopy of single crystals of camphor- bound P450CAM in the oxidized, reduced, and CO-reduced states, and iterative extended Ruckel (IEH) calculations. The heme chromophores are orientated such that transitions polarized in the heme plane (x,y-polarized) can be readily distinguished from transitions polarized perpendicular to the heme plane (z-polarized) . High spin oxidized P450 exhibits two broad z-polarized bands, at 567 and 323 nm. IEH calculations suggest that these bands arise from cysteine mercaptide sulfur-to-iron charge transfer transitions. High spin reduced P450 has no z-polarized bands. IEH calculations suggest that loss of these bands occurs because the cysteine sulfur is protonated to a mercaptan. Low spin CO-P450 has an intense x,y-polarized band at 363 nm. This transition, assigned as a mercaptide sulfur-to-porphyrin charge transfer transition, has the correct symmetry to mix with the Soret and may cause the anomalous red shift of the Soret

Dynamics of Electron Transport in Cytochrome P450 Systems Studied at Sub-Zero Temperature

Experimentation in fluid mixed solvents (1 : 1 v/v phosphate buffer ethylene glycol) at sub-zero temperatures has permitted us to record the two univalent reductions of the bacterial cytochrome P450 by the natural electron donor putidaredoxin, without recycling or alternative pathway reactions. Dynamic evidence shows the formation of putidaredoxincytochrome complexes prior to electron transfer. The complex formation is rate limiting in the first reduction and in our experimental conditions. The kinetics of binding between the two oxidized proteins has also been recorded in the same medium under various conditions of concentration, temperature and ionic strength. At very low ionic strength, the rate is limited by electrostatic repulsion between the two negatively charge proteins; above I = 0.03 this effect appears negligible and the affinity seems to be governed by hydrophobic interaction free energy

Dynamics of Electron Transport in Cytochrome P450 Systems Studied at Sub-Zero Temperature

Experimentation in fluid mixed solvents (1 : 1 v/v phosphate buffer ethylene glycol) at sub-zero temperatures has permitted us to record the two univalent reductions of the bacterial cytochrome P450 by the natural electron donor putidaredoxin, without recycling or alternative pathway reactions. Dynamic evidence shows the formation of putidaredoxincytochrome complexes prior to electron transfer. The complex formation is rate limiting in the first reduction and in our experimental conditions. The kinetics of binding between the two oxidized proteins has also been recorded in the same medium under various conditions of concentration, temperature and ionic strength. At very low ionic strength, the rate is limited by electrostatic repulsion between the two negatively charge proteins; above I = 0.03 this effect appears negligible and the affinity seems to be governed by hydrophobic interaction free energy

Protein components of a cytochrome P-450 linalool 8-methyl hydroxylase

The cytochrome P-450 heme-thiolate monooxygenases that hydroxylate monoterpene hydrocarbon groups are effective models for the cytochrome P-450 family. We have purified and characterized the three proteins from a P-450-dependent linalool 8-methyl hydroxylase in Pseudomonas putida (incognita) strain PpG777. The proteins resemble the camphor 5-exohydroxylase components in chemical and physical properties; however, they show neither immunological cross-reactivity nor catalytic activity in heterogenous recombination. These two systems provide an excellent model to probe more deeply the heme-thiolate reaction center, molecular domains of substrate specificity, redox-pair interactions, and the regulation of the reaction cycle

Electronic Structure of Cytochrome P450

The optical properties of P450 have been investigated by means of polarized absorption spectroscopy of single crystals of camphor- bound P450CAM in the oxidized, reduced, and CO-reduced states, and iterative extended Ruckel (IEH) calculations. The heme chromophores are orientated such that transitions polarized in the heme plane (x,y-polarized) can be readily distinguished from transitions polarized perpendicular to the heme plane (z-polarized) . High spin oxidized P450 exhibits two broad z-polarized bands, at 567 and 323 nm. IEH calculations suggest that these bands arise from cysteine mercaptide sulfur-to-iron charge transfer transitions. High spin reduced P450 has no z-polarized bands. IEH calculations suggest that loss of these bands occurs because the cysteine sulfur is protonated to a mercaptan. Low spin CO-P450 has an intense x,y-polarized band at 363 nm. This transition, assigned as a mercaptide sulfur-to-porphyrin charge transfer transition, has the correct symmetry to mix with the Soret and may cause the anomalous red shift of the Soret

Boolean network model predicts cell cycle sequence of fission yeast

A Boolean network model of the cell-cycle regulatory network of fission yeast (Schizosaccharomyces Pombe) is constructed solely on the basis of the known biochemical interaction topology. Simulating the model in the computer, faithfully reproduces the known sequence of regulatory activity patterns along the cell cycle of the living cell. Contrary to existing differential equation models, no parameters enter the model except the structure of the regulatory circuitry. The dynamical properties of the model indicate that the biological dynamical sequence is robustly implemented in the regulatory network, with the biological stationary state G1 corresponding to the dominant attractor in state space, and with the biological regulatory sequence being a strongly attractive trajectory. Comparing the fission yeast cell-cycle model to a similar model of the corresponding network in S. cerevisiae, a remarkable difference in circuitry, as well as dynamics is observed. While the latter operates in a strongly damped mode, driven by external excitation, the S. pombe network represents an auto-excited system with external damping.Comment: 10 pages, 3 figure

The iron electron-nuclear double resonance (ENDOR) of two-iron ferredoxins from spinach, parsley, pig adrenal cortex and Pseudomonas putida

The iron electron-nuclear double resonance (ENDOR) spectra of reduced iron-sulfur proteins (two-iron ferredoxins) from spinach, parsley, pig adrenal cortex and Pseudomonas putida unequivocally show two inequivalent iron atoms at the active sites of each of these proteins. The frequencies of the ENDOR lines establish the total electronic spin in the ground state to be . The principal values of the hyperfine tensor have been determined for each of the iron atoms and these values are consistent with and lend considerable support to the model of a high-spin Fe(III) atom and a high-spin Fe(II) atom antiferromagnetically coupled to form an system. The measured principal axis components of the effective hyperfine tensors for are as follows (1 and 2 refer to the inequivalent iron sites): These data are consistent with Site 1 being ferric and Site 2, ferrous iron. The primes indicate that the A-tensor principal axes for Site 1 (Fe(III)) are apparently rotated about the x-axis with respect to the g-tensor axes by an angle [theta] (20[deg] [les] [theta] [les] 40[deg]). The orientations of the A-tensors for Site 2 (Fe(II)) have not been determined and hence the values presented are the observed values of the A-tensors along the x, y, and z-axes of the g-tensor for this complex.A brief introduction to the theory of ENDOR is given.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33533/1/0000032.pd

EcoCyc: A comprehensive view of Escherichia coli biology

EcoCyc (http://EcoCyc.org) provides a comprehensive encyclopedia of Escherichia coli biology. EcoCyc integrates information about the genome, genes and gene products; the metabolic network; and the regulatory network of E. coli. Recent EcoCyc developments include a new initiative to represent and curate all types of E. coli regulatory processes such as attenuation and regulation by small RNAs. EcoCyc has started to curate Gene Ontology (GO) terms for E. coli and has made a dataset of E. coli GO terms available through the GO Web site. The curation and visualization of electron transfer processes has been significantly improved. Other software and Web site enhancements include the addition of tracks to the EcoCyc genome browser, in particular a type of track designed for the display of ChIP-chip datasets, and the development of a comparative genome browser. A new Genome Omics Viewer enables users to paint omics datasets onto the full E. coli genome for analysis. A new advanced query page guides users in interactively constructing complex database queries against EcoCyc. A Macintosh version of EcoCyc is now available. A series of Webinars is available to instruct users in the use of EcoCyc

EcoCyc: a comprehensive database of Escherichia coli biology

EcoCyc (http://EcoCyc.org) is a comprehensive model organism database for Escherichia coli K-12 MG1655. From the scientific literature, EcoCyc captures the functions of individual E. coli gene products; their regulation at the transcriptional, post-transcriptional and protein level; and their organization into operons, complexes and pathways. EcoCyc users can search and browse the information in multiple ways. Recent improvements to the EcoCyc Web interface include combined gene/protein pages and a Regulation Summary Diagram displaying a graphical overview of all known regulatory inputs to gene expression and protein activity. The graphical representation of signal transduction pathways has been updated, and the cellular and regulatory overviews were enhanced with new functionality. A specialized undergraduate teaching resource using EcoCyc is being developed

FORG3D: Force-directed 3D graph editor for visualization of integrated genome scale data

<p>Abstract</p> <p>Background</p> <p>Genomics research produces vast amounts of experimental data that needs to be integrated in order to understand, model, and interpret the underlying biological phenomena. Interpreting these large and complex data sets is challenging and different visualization methods are needed to help produce knowledge from the data.</p> <p>Results</p> <p>To help researchers to visualize and interpret integrated genomics data, we present a novel visualization method and bioinformatics software tool called FORG3D that is based on real-time three-dimensional force-directed graphs. FORG3D can be used to visualize integrated networks of genome scale data such as interactions between genes or gene products, signaling transduction, metabolic pathways, functional interactions and evolutionary relationships. Furthermore, we demonstrate its utility by exploring gene network relationships using integrated data sets from a <it>Caenorhabditis elegans </it>Parkinson's disease model.</p> <p>Conclusion</p> <p>We have created an open source software tool called FORG3D that can be used for visualizing and exploring integrated genome scale data.</p