592 resultados para Histidine


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Mycobacterium tuberculosis genes Rv0844c/Rv0845 encoding the NarL response regulator and NarS histidine kinase are hypothesized to constitute a two-component system involved in the regulation of nitrate metabolism. However, there is no experimental evidence to support this. In this study, we established M. tuberculosis NarL/NarS as a functional two-component system and identified His(241) and Asp(61) as conserved phosphorylation sites in NarS and NarL, respectively. Transcriptional profiling between M. tuberculosis H37Rv and Delta narL mutant strain during exponential growth in broth cultures with or without nitrate defined an similar to 30-gene NarL regulon that exhibited significant overlap with DevR-regulated genes, thereby implicating a role for the DevR response regulator in the regulation of nitrate metabolism. Notably, expression analysis of a subset of genes common to NarL and DevR regulons in M. tuberculosis Delta devR, Delta devS Delta dosT, and Delta narL mutant strains revealed that in response to nitrite produced during aerobic nitrate metabolism, the DevRS/DosT regulatory system plays a primary role that is augmented by NarL. Specifically, NarL itself was unable to bind to the narK2, acg, and Rv3130c promoters in phosphorylated or unphosphorylated form; however, its interaction with DevR similar to P resulted in cooperative binding, thereby enabling co-regulation of these genes. These findings support the role of physiologically derived nitrite as a metabolic signal in mycobacteria. We propose NarL-DevR binding, possibly as a heterodimer, as a novel mechanism for co-regulation of gene expression by the DevRS/DosT and NarL/NarS regulatory systems.

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Background: Computational protein design is a rapidly maturing field within structural biology, with the goal of designing proteins with custom structures and functions. Such proteins could find widespread medical and industrial applications. Here, we have adapted algorithms from the Rosetta software suite to design much larger proteins, based on ideal geometric and topological criteria. Furthermore, we have developed techniques to incorporate symmetry into designed structures. For our first design attempt, we targeted the (alpha/beta)(8) TIM barrel scaffold. We gained novel insights into TIM barrel folding mechanisms from studying natural TIM barrel structures, and from analyzing previous TIM barrel design attempts. Methods: Computational protein design and analysis was performed using the Rosetta software suite and custom scripts. Genes encoding all designed proteins were synthesized and cloned on the pET20-b vector. Standard circular dichroism and gel chromatographic experiments were performed to determine protein biophysical characteristics. 1D NMR and 2D HSQC experiments were performed to determine protein structural characteristics. Results: Extensive protein design simulations coupled with ab initio modeling yielded several all-atom models of ideal, 4-fold symmetric TIM barrels. Four such models were experimentally characterized. The best designed structure (Symmetrin-1) contained a polar, histidine-rich pore, forming an extensive hydrogen bonding network. Symmetrin-1 was easily expressed and readily soluble. It showed circular dichroism spectra characteristic of well-folded alpha/beta proteins. Temperature melting experiments revealed cooperative and reversible unfolding, with a T-m of 44 degrees C and a Gibbs free energy of unfolding (Delta G degrees) of 8.0 kJ/mol. Urea denaturing experiments confirmed these observations, revealing a C-m of 1.6 M and a Delta G degrees of 8.3 kJ/mol. Symmetrin-1 adopted a monomeric conformation, with an apparent molecular weight of 32.12 kDa, and displayed well resolved 1D-NMR spectra. However, the HSQC spectrum revealed somewhat molten characteristics. Conclusions: Despite the detection of molten characteristics, the creation of a soluble, cooperatively folding protein represents an advancement over previous attempts at TIM barrel design. Strategies to further improve Symmetrin-1 are elaborated. Our techniques may be used to create other large, internally symmetric proteins.

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A lectin from phloem exudates of Luffa acutangula (ridge gourd) was purified on chitin affinity chromatography and characterized for its amino acid sequence and to study the role of tryptophan in its activity. The purified lectin was subjected to various proteolytic digestions, and the resulting peptides were analyzed by liquid chromatography coupled electrospray ionization ion trap mass spectrometer. The peptide precursor ions were fragmented by collision-induced dissociation or electron transfer dissociation experiments, and a manual interpretation of MS/MS was performed to deduce amino acid sequence. This gave rise to almost complete sequence coverage of the lectin which showed high-sequence similarity with deduced sequences of phloem lectins present in the database. Chemical modification of lysine, tyrosine, histidine, arginine, aspartic acid, and glutamic acid residues did not inhibit the hemagglutinating activity. However, the modification of tryptophan residues using N-bromosuccinimide showed the loss of hemagglutinating activity. Additionally, the mapping of tryptophan residues was performed to determine the extent and number of residues modified, which revealed that six residues per molecule were oxidized suggesting their accessibility. The retention of the lectin activity was seen when the modifications were performed in the presence of chitooligosaccharides due to protection of a tryptophan residue (W-102) in the protein. These studies taken together have led to the identification of a particular tryptophan residue (W-102) in the activity of the lectin. (c) 2015 IUBMB Life, 67(12):943-953, 2015

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Molecular dynamics (MD) simulation is employed to study the bio-adhesion in F1 ATP molecular motor. Histidine-peptide is widely used as linkage in micro systems because of its strong binding strength to metals. This paper focuses on the adhesion between a synthetic peptide containing 6xHis-tag (Gly-Gly-Lys-Gly-Gly-Lys-Gly-Gly-His-His-His-His-His-His) and metal substrate, which is used to define the position of the F1 ATP molecular motor on the metal substrate. It is shown that the binding strength between histidine and nickel substrate is the strongest, while that of copper is smaller and that of gold substrate is the smallest. From the result of simulation, we find that the stability of adhesion between histidine and the metal substate result of the ringed structure in histidine.

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The design, synthesis, and characterization of two novel metalloprotein motifs is presented. The first project involved the design and construction of a protein motif which was programmed to form a tetradentate metal complex upon the addition of metal cations. The overall structure of the motif was based on a ββ super-secondary structure consisting of a flexible peptide sequence flanked by metal binding regions located at the carboxy and amino termini. The metal binding region near the amino terminus was constructed from a reverse turn motif with two metal ligating residues, (2R, 3R)-β-methyl-cysteine and histidine. Selection of the peptide sequence for this region was based on the conformational analysis of a series of tetrapeptides designed to form reverse turns in solution.

The stereospecific syntheses of a series of novel bipyridyl- and phenanthrolylsubstituted amino acids was carried out to provide ligands for the carboxy terminus metal binding region. These residues were incorporated into peptide sequences using solid phase peptide synthesis protocols, and metal binding studies indicated that the metal binding properties of these ligands was dictated by the specific regioisomer of the heteroaromatic ring and the peptide primary sequence.

Finally, a peptide containing optimized components for the metal binding regions was prepared to test the ability of the compound to form the desired intramolecular peptide:metal cation complexes. Metal binding studies demonstrated that the peptide formed monomeric complexes with very high metal cation binding affinities and that the two metal binding regions act cooperatively in the metal binding process. The use of these systems in the design of proteins capable of regulating naturally occurring proteins is discussed.

The second project involved the semisynthesis of two horse heart cytochrome c mutants incorporating the bipyridyl-amino acids at position 72 of the protein sequence. Structural studies on the proteins indicated that the bipyridyl amino acids had a neglible effect on the protein structure. One of the mutants was modified with Ru(bpy)_2^(+2) to form a redox-active protein, and the modified protein was found to have enhanced electron transfer properties between the heme and the introduced metal site.

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This thesis describes research pursued in two areas, both involving the design and synthesis of sequence specific DNA-cleaving proteins. The first involves the use of sequence-specific DNA-cleaving metalloproteins to probe the structure of a protein-DNA complex, and the second seeks to develop cleaving moieties capable of DNA cleavage through the generation of a non-diffusible oxidant under physiological conditions.

Chapter One provides a brief review of the literature concerning sequence-specific DNA-binding proteins. Chapter Two summarizes the results of affinity cleaving experiments using leucine zipper-basic region (bZip) DNA-binding proteins. Specifically, the NH_2-terminal locations of a dimer containing the DNA binding domain of the yeast transcriptional activator GCN4 were mapped on the binding sites 5'-CTGACTAAT-3' and 5'ATGACTCTT- 3' using affinity cleaving. Analysis of the DNA cleavage patterns from Fe•EDTA-GCN4(222-281) and (226-281) dimers reveals that the NH_2-termini are in the major groove nine to ten base pairs apart and symmetrically displaced four to five base pairs from the central C of the recognition site. These data are consistent with structural models put forward for this class of DNA binding proteins. The results of these experiments are evaluated in light of the recently published crystal structure for the GCN4-DNA complex. Preliminary investigations of affinity cleaving proteins based on the DNA-binding domains of the bZip proteins Jun and Fos are also described.

Chapter Three describes experiments demonstrating the simultaneous binding of GCN4(226-281) and 1-Methylimidazole-2-carboxamide-netropsin (2-ImN), a designed synthetic peptide which binds in the minor groove of DNA at 5'-TGACT-3' sites as an antiparallel, side-by-side dimer. Through the use of Fe•EDTA-GCN4(226-281) as a sequence-specific footprinting agent, it is shown that the dimeric protein GCN4(226-281) and the dimeric peptide 2- ImN can simultaneously occupy their common binding site in the major and minor grooves of DNA, respectively. The association constants for 2-ImN in the presence and in the absence of Fe•EDTA-GCN4(226-281) are found to be similar, suggesting that the binding of the two dimers is not cooperative.

Chapter Four describes the synthesis and characterization of PBA-β-OH-His- Hin(139-190), a hybrid protein containing the DNA-binding domain of Hin recombinase and the putative iron-binding and oxygen-activating domain of the antitumor antibiotic bleomycin. This 54-residue protein, comprising residues 139-190 of Hin recombinase with the dipeptide pyrimidoblamic acid-β-hydroxy-L-histidine (PBA-β-OH-His) at the NH2 terminus, was synthesized by solid phase methods. PBA-β-OH-His-Hin(139- 190) binds specifically to DNA at four distinct Hin binding sites with affinities comparable to those of the unmodified Hin(139-190). In the presence of dithiothreitol (DTT), Fe•PB-β-OH-His-Hin(139-190) cleaves DNA with specificity remarkably similar to that of Fe•EDTA-Hin(139-190), although with lower efficiency. Analysis of the cleavage pattern suggests that DNA cleavage is mediated through a diffusible species, in contrast with cleavage by bleomycin, which occurs through a non-diffusible oxidant.

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Redox-active ruthenium complexes have been covalently attached to the surface of a series of natural, semisynthetic and recombinant cytochromes c. The protein derivatives were characterized by a variety of spectroscopic techniques. Distant Fe^(2+) - Ru^(3+) electronic couplings were extracted from intramolecular electron-transfer rates in Ru(bpy)_2(im)HisX (where X= 33, 39, 62, and 72) derivatives of cyt c. The couplings increase according to 62 (0.0060) < 72 (0.057) < 33 (0.097) < 39 (0.11 cm^(-1)); however, this order is incongruent with histidine to heme edge-edge distances [62 (14.8) > 39 (12.3) > 33 (11.1) > =72 (8.4 Å)]. These results suggest the chemical nature of the intervening medium needs to be considered for a more precise evaluation of couplings. The rates (and couplings) correlate with the lengths of a-tunneling pathways comprised of covalent bonds, hydrogen bonds and through-space jumps from the histidines to the heme group. Space jumps greatly decrease couplings: one from Pro71 to Met80 extends the σ-tunneling length of the His72 pathway by roughly 10 covalent bond units. Experimental couplings also correlate well with those calculated using extended Hiickel theory to evaluate the contribution of the intervening protein medium.

Two horse heart cyt c variants incorporating the unnatural amino acids (S)-2- amino-3-(2,2'-bipyrid-6-yl)-propanoic acid (6Bpa) and (S)-2-amino-3-(2,2'-bipyrid-4-yl)propanoic acid ( 4Bpa) at position 72 have been prepared using semisynthetic protocols. Negligible perturbation of the protein structure results from this introduction of unnatural amino acids. Redox-active Ru(2,2'-bipyridine)_2^(2+) binds to 4Bpa72 cyt c but not to the 6Bpa protein. Enhanced ET rates were observed in the Ru(bpy)_2^(2+)-modified 4Bpa72 cyt c relative to the analogous His72 derivative. The rapid (< 60 nanosecond) photogeneration of ferrous Ru-modified 4Bpa72 cyt c in the conformationally altered alkaline state demonstrates that laser-induced ET can be employed to study submicrosecond protein-folding events.

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β-lactamases are a group of enzymes that confer resistance to penam and cephem antibiotics by hydrolysis of the β-lactam ring, thereby inactivating the antibiotic. Crystallographic and computer modeling studies of RTEM-1 β-lactamase have indicated that Asp 132, a strictly conserved residue among the class A β-lactamases, appears to be involved in substrate binding, catalysis, or both. To study the contribution of residue 132 to β-lactamase function, site saturation mutagenesis was used to generate mutants coding for all 20 amino acids at position 132. Phenotypic screening of all mutants indicated that position 132 is very sensitive to amino acid changes, with only N132C, N132D, N132E, and N132Q showing any appreciable activity. Kinetic analysis of three of these mutants showed increases in K_M, along with substantial decreases in k_(cat). Efforts to trap a stable acyl-enzyme intermediate were unsuccessfuL These results indicate that residue 132 is involved in substrate binding, as well as catalysis, and supports the involvement of this residue in acylation as suggested by Strynadka et al.

Crystallographic and computer modeling studies of RTEM-1 β-lactamase have indicated that Lys 73 and Glu 166, two strictly conserved residues among the class A β-lactamases, appear to be involved in substrate binding, catalysis, or both. To study the contribution of these residues to β-lactamase function, site saturation mutagenesis was used to generate mutants coding for all 20 amino acids at positions 73 and 166. Then all 400 possible combinations of mutants were created by combinatorial mutagenesis. The colonies harboring the mutants were screened for growth in the presence of ampicillin. The competent colonys' DNA were sequenced, and kinetic parameters investigated. It was found that lysine is essential at position 73, and that position 166 only tolerated fairly conservative changes (Aspartic acid, Histidine, and Tyrosine). These functional mutants exhibited decreased kcat's, but K_M was close to wild-type levels. The results of the combinatorial mutagenesis experiments indicate that Lysis absolutely required for activity at position 73; no mutation at residue 166 can compensate for loss of the long side chain amine. The active mutants found--K73K/E166D, K73KIE166H, and K73KIE166Y were studied by kinetic analysis. These results reaffirmed the function of residue 166 as important in catalysis, specifically deacylation.

The identity of the residue responsible for enhancing the active site serine (Ser 70) in RTEM-1 β-lactamase has been disputed for some time. Recently, analysis of a crystal structure of RTEM-1 β-lactamase with covalently bound intermediate was published, and it was suggested that Lys 73, a strictly conserved residue among the class A β-lactamases, was acting as a general base, activating Ser 70. For this to be possible, the pK_a of Lys 73 would have to be depressed significantly. In an attempt to assay the pK_a of Lys 73, the mutation K73C was made. This mutant protein can be reacted with 2-bromoethylamine, and activity is restored to near wild type levels. ^(15)N-2-bromoethylamine hydrobromide and ^(13)C-2-bromoethylamine hydrobromide were synthesized. Reacting these compounds with the K73C mutant gives stable isotopic enrichment at residue 73 in the form of aminoethylcysteine, a lysine homologue. The pK_a of an amine can be determined by NMR titration, following the change in chemical shift of either the ^(15)N-amine nuclei or adjacent Be nuclei as pH is changed. Unfortunately, low protein solubility, along with probable label scrambling in the Be experiment, did not permit direct observation of either the ^(15)N or ^(13)C signals. Indirect detection experiments were used to observe the protons bonded directly to the ^(13)C atoms. Two NMR signals were seen, and their chemical shift change with pH variation was noted. The peak which was determined to correspond to the aminoethylcysteine residue shifted from 3.2 ppm down to 2.8 ppm over a pH range of 6.6 to 12.5. The pK_a of the amine at position 73 was determined to be ~10. This indicates that residue 73 does not function as a general base in the acylation step of the reaction. However the experimental measurement takes place in the absence of substrate. Since the enzyme undergoes conformational changes upon substrate binding, the measured pK_a of the free enzyme may not correspond to the pK_a of the enzyme substrate complex.

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Multi-step electron tunneling, or “hopping,” has become a fast-developing research field with studies ranging from theoretical modeling systems, inorganic complexes, to biological systems. In particular, the field is exploring hopping mechanisms in new proteins and protein complexes, as well as further understanding the classical biological hopping systems such as ribonuclease reductase, DNA photolyases, and photosystem II. Despite the plethora of natural systems, only a few biologically engineered systems exist. Engineered hopping systems can provide valuable information on key structural and electronic features, just like other kinds of biological model systems. Also, engineered systems can harness common biologic processes and utilize them for alternative reactions. In this thesis, two new hopping systems are engineered and characterized.

The protein Pseudomonas aeruginosa azurin is used as a building block to create the two new hopping systems. Besides being well studied and amenable to mutation, azurin already has been used to successfully engineer a hopping system. The two hopping systems presented in this thesis have a histidine-attached high potential rhenium 4,7-dimethyl-1,10-phenanthroline tricarbonyl [Re(dmp)(CO)3] + label which, when excited, acts as the initial electron acceptor. The metal donor is the type I copper of the azurin protein. The hopping intermediates are all tryptophan, an amino acid mutated into the azurin at select sites between the photoactive metal label and the protein metal site. One system exhibits an inter-molecular hopping through a protein dimer interface; the other system undergoes intra-molecular multi-hopping utilizing a tryptophan “wire.” The electron transfer reactions are triggered by excitation of the rhenium label and monitored by UV-Visible transient absorption, luminescence decays measurements, and time-resolved Infrared spectroscopy (TRIR). Both systems were structurally characterized by protein X-ray crystallography.

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A study of the pH and temperature dependence of the redox potentials of azurins from five species of bacteria has been performed. The variations in the potentials with pH have been interpreted in terms of electrostatic interactions between the copper site and titrating histidine residues, including the effects of substitutions in the amino acid sequences of the proteins on the electrostatic interactions. A comparison of the observed pH dependences with predictions based on histidine pK_a values known for Pseudomonas aeruginosa (Pae), Alcaligenes denitrificans (Ade), and Alcaligenes faecalis (Afa) azurins indicates that the Pae and Ade redox potentials exhibit pH dependences in line with electrostatic arguments, while Afa azurin exhibits more complex behavior. Redox enthalpies and entropies for four of the azurins at low and high pH values have also been obtained. Based on these results in conjuction with the variable pH experiments, it appears that Bordetella bronchiseptica azurin may undergo a more substantial conformational change with pH than has been observed for other species of azurin.

The temperature dependence of the redox potential of bovine erythrocyte superoxide dismutase (SOD) has been determined at pH 7.0, with potassium ferricyanide as the mediator. The following thermodynamic parameters have been obtained (T = 25°C): E°' = 403±5 mV vs. NHE, ΔG°' = -9.31 kcal/mol, ΔH°' = -21.4 kcal/mol, ΔS°' = -40.7 eu, ΔS°'_(rc) = -25.1 eu. It is apparent from these results that ΔH°', rather than ΔS°', is the dominant factor in establishing the high redox potential of SOD. The large negative enthalpy of reduction may also reflect the factors which give SOD its high specificity toward reduction and oxidation by superoxide.

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A semisynthetic binuclear metalloprotein has been prepared by appending the pentaammineruthenium moiety to histidine 39 of the cytochrome c from the yeast Candida krusei. The site of ruthenium binding was identified by peptide mapping. Spectroscopic and electrochemical properties of the derivative indicate the protein conformation is unperturbed by the modification. A preliminary (minimum) rate constant of 170s^(-1) has been determined for the intramolecular electron transfer from ruthenium(II) to iron(III), which occurs over a distance of at least 13Å (barring major conformational changes). Electrochemical studies indicate that this reaction should proceed with a driving force of ~170mV. The rate constant is an order of magnitude faster than that observed in horse heart cytochrome c for intramolecular electron transfer from pentaammineruthenium(II)(histidine 33) to iron(III) (over a similar distance, and with a similar driving force), suggesting a medium or orientation effect makes the Candida intramolecular electron transfer more favorable.

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Because so little is known about the structure of membrane proteins, an attempt has been made in this work to develop techniques by which to model them in three dimensions. The procedures devised rely heavily upon the availability of several sequences of a given protein. The modelling procedure is composed of two parts. The first identifies transmembrane regions within the protein sequence on the basis of hydrophobicity, β-turn potential, and the presence of certain amino acid types, specifically, proline and basic residues. The second part of the procedure arranges these transmembrane helices within the bilayer based upon the evolutionary conservation of their residues. Conserved residues are oriented toward other helices and variable residues are positioned to face the surrounding lipids. Available structural information concerning the protein's helical arrangement, including the lengths of interhelical loops, is also taken into account. Rhodopsin, band 3, and the nicotinic acetylcholine receptor have all been modelled using this methodology, and mechanisms of action could be proposed based upon the resulting structures.

Specific residues in the rhodopsin and iodopsin sequences were identified, which may regulate the proteins' wavelength selectivities. A hinge-like motion of helices M3, M4, and M5 with respect to the rest of the protein was proposed to result in the activation of transducin, the G-protein associated with rhodopsin. A similar mechanism is also proposed for signal transduction by the muscarinic acetylcholine and β-adrenergic receptors.

The nicotinic acetylcholine receptor was modelled with four trans-membrane helices per subunit and with the five homologous M2 helices forming the cation channel. Putative channel-lining residues were identified and a mechanism of channel-opening based upon the concerted, tangential rotation of the M2 helices was proposed.

Band 3, the anion exchange protein found in the erythrocyte membrane, was modelled with 14 transmembrane helices. In general the pathway of anion transport can be viewed as a channel composed of six helices that contains a single hydrophobic restriction. This hydrophobic region will not allow the passage of charged species, unless they are part of an ion-pair. An arginine residue located near this restriction is proposed to be responsible for anion transport. When ion-paired with a transportable anion it rotates across the barrier and releases the anion on the other side of the membrane. A similar process returns it to its original position. This proposed mechanism, based on the three-dimensional model, can account for the passive, electroneutral, anion exchange observed for band 3. Dianions can be transported through a similar mechanism with the additional participation of a histidine residue. Both residues are located on M10.

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Semisynthesis of horse heart cytochrome c and site-directed mutagenesis of Saccharomyces cerevisiae (S. c.) iso-1-cytochrome c have been utilized to substitute Ala for the cytochrome c heme axial ligand Met80 to yield ligand-binding proteins (horse heart Ala80cyt c and S.c. Ala80cyt c) with spectroscopic properties remarkably similar to those of myoglobin. Both species of Fe(II)Ala80cyt c form exceptionally stable dioxygen complexes with autoxidation rates 10-30x smaller and O2 binding constants ~ 3x greater than those of myoglobin. The resistance of O2-Fe(II)Ala80cyt c to autoxidation is attributed in part to protection of the heme site from solvent as exhibited by the exceptionally slow rate of CO binding to the heme as well as the low quantum yield of CO photodissociation.

UV/vis, EPR, and paramagnetic NMR spectroscopy indicate that at pH 7 the Fe(III)Ala80cyt c heme is low-spin with axial His-OH- coordination and that below pH ~6.5, Fe(III)Ala80cyt cis high-spin with His-H2O heme ligation. Significant differences in the pH dependence of the 1H NMR spectra of S.c. Fe(III)Ala80cyt c compared to wild-type demonstrate that the axial ligands influence the conformational energetics of cytochrome c.

1H NMR spectroscopy has been utilized to determine the solution structure of the cyanide derivative of S.c. Fe(III)Ala80cyt c. 82% of the resonances in the 1H NMR spectrum of S.c. CN-Fe(III)Ala80cyt c have been assigned through 1D and 2D experiments. The RMSD values after restrained energy minimization of the family of 17 structures obtained from distance geometry calculations are 0.68 ± 0.11 Å for the backbone and 1.32 ± 0.14 Å for all heavy atoms. The solution structure indicates that a tyrosine in the "distal" pocket of CN-Fe(III)Ala80cyt c forms a hydrogen bond with the Fe(III)-CN unit, suggesting that it may play a role analogous to that of the distal histidine in myoglobin in stabilizing the dioxygen adduct.

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A summary of previous research is presented that indicates that the purpose of a blue copper protein's fold and hydrogen bond network, aka, the rack effect, enforce a copper(II) geometry around the copper(I) ion in the metal site. In several blue copper proteins, the C-terminal histidine ligand becomes protonated and detaches from the copper in the reduced forms. Mutants of amicyanin from Paracoccus denitrificans were made to alter the hydrogen bond network and quantify the rack effect by pKa shifts.

The pKa's of mutant amicyanins have been measured by pH-dependent electrochemistry. P94F and P94A mutations loosen the Northern loop, allowing the reduced copper to adopt a relaxed conformation: the ability to relax drives the reduction potentials up. The measured potentials are 265 (wild type), 380 (P94A), and 415 (P94F) mV vs. NHE. The measured pKa's are 7.0 (wild type), 6.3 (P94A), and 5.0 (P94F). The additional hydrogen bond to the thiolate in the mutants is indicated by a red-shift in the blue copper absorption and an increase in the parallel hyperfine splitting in the EPR spectrum. This hydrogen bond is invoked as the cause for the increased stability of the C-terminal imidazole.

Melting curves give a measure of the thermal stability of the protein. A thermodynamic intermediate with pH-dependent reversibility is revealed. Comparisons with the electrochemistry and apoamicyanin suggest that the intermediate involves the region of the protein near the metal site. This region is destabilized in the P94F mutant; coupled with the evidence that the imidazole is stabilized under the same conditions confirms an original concept of the rack effect: a high energy configuration is stabilized at a cost to the rest of the protein.

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The cytochromes P450 (P450s) are a remarkable class of heme enzymes that catalyze the metabolism of xenobiotics and the biosynthesis of signaling molecules. Controlled electron flow into the thiolate-ligated heme active site allows P450s to activate molecular oxygen and hydroxylate aliphatic C–H bonds via the formation of high-valent metal-oxo intermediates (compounds I and II). Due to the reactive nature and short lifetimes of these intermediates, many of the fundamental steps in catalysis have not been observed directly. The Gray group and others have developed photochemical methods, known as “flash-quench,” for triggering electron transfer (ET) and generating redox intermediates in proteins in the absence of native ET partners. Photo-triggering affords a high degree of temporal precision for the gating of an ET event; the initial ET and subsequent reactions can be monitored on the nanosecond-to-second timescale using transient absorption (TA) spectroscopies. Chapter 1 catalogues critical aspects of P450 structure and mechanism, including the native pathway for formation of compound I, and outlines the development of photochemical processes that can be used to artificially trigger ET in proteins. Chapters 2 and 3 describe the development of these photochemical methods to establish electronic communication between a photosensitizer and the buried P450 heme. Chapter 2 describes the design and characterization of a Ru-P450-BM3 conjugate containing a ruthenium photosensitizer covalently tethered to the P450 surface, and nanosecond-to-second kinetics of the photo-triggered ET event are presented. By analyzing data at multiple wavelengths, we have identified the formation of multiple ET intermediates, including the catalytically relevant compound II; this intermediate is generated by oxidation of a bound water molecule in the ferric resting state enzyme. The work in Chapter 3 probes the role of a tryptophan residue situated between the photosensitizer and heme in the aforementioned Ru-P450 BM3 conjugate. Replacement of this tryptophan with histidine does not perturb the P450 structure, yet it completely eliminates the ET reactivity described in Chapter 2. The presence of an analogous tryptophan in Ru-P450 CYP119 conjugates also is necessary for observing oxidative ET, but the yield of heme oxidation is lower. Chapter 4 offers a basic description of the theoretical underpinnings required to analyze ET. Single-step ET theory is first presented, followed by extensions to multistep ET: electron “hopping.” The generation of “hopping maps” and use of a hopping map program to analyze the rate advantage of hopping over single-step ET is described, beginning with an established rhenium-tryptophan-azurin hopping system. This ET analysis is then applied to the Ru-tryptophan-P450 systems described in Chapter 2; this strongly supports the presence of hopping in Ru-P450 conjugates. Chapter 5 explores the implementation of flash-quench and other phototriggered methods to examine the native reductive ET and gas binding events that activate molecular oxygen. In particular, TA kinetics that demonstrate heme reduction on the microsecond timescale for four Ru-P450 conjugates are presented. In addition, we implement laser flash-photolysis of P450 ferrous–CO to study the rates of CO rebinding in the thermophilic P450 CYP119 at variable temperature. Chapter 6 describes the development and implementation of air-sensitive potentiometric redox titrations to determine the solution reduction potentials of a series of P450 BM3 mutants, which were designed for non-native cyclopropanation of styrene in vivo. An important conclusion from this work is that substitution of the axial cysteine for serine shifts the wild type reduction potential positive by 130 mV, facilitating reduction by biological redox cofactors in the presence of poorly-bound substrates. While this mutation abolishes oxygenation activity, these mutants are capable of catalyzing the cyclopropanation of styrene, even within the confines of an E. coli cell. Four appendices are also provided, including photochemical heme oxidation in ruthenium-modified nitric oxide synthase (Appendix A), general protocols (Appendix B), Chapter-specific notes (Appendix C) and Matlab scripts used for data analysis (Appendix D).