Electron transfer in cytochrome c oxidase : the rate of electron equillibration between cytochrome a and copper A

Intramolecular electron transfer in partially reduced cytochrome c oxidase has been studied by means of perturbed equilibrium techniques. We have prepared a three electron reduced, CO inhibited form of the enzyme in which cytochrome a and copper A are partially reduced an in intramolecular redox equilibrium. When these samples were photolyzed using a nitrogen laser (0.6 µs, 1.0 mJ pulses) changes in absorbance at 598 nm and 830 nm were observed which are consistent with a fast electron from cytochrome a to copper A. The absorbance changes at 598 nm have an apparent rate of 17,200 ± 1,700 s^(-1) (1σ), at pH 7.0 and 25.5 °C. These changes were not observed in either the CO mixed valence or CO inhibited fully reduced forms of the enzyme. The rate is fastest at about pH 8.0, and falls off in either direction, and there is a small, but clear temperature dependence. The process was also observed in the cytochrome c -- cytochrome c oxidase high affinity complex.

This rate is far faster than any rate measured or inferred previously for the cytochrome a -- copper A electron equilibration, but the interpretation of these results is hampered by the fact that the relaxation could only be followed during the time before CO became rebound to the oxygen binding site. The meaning of our our measured rate is discussed, along with other reported rates for this process. In addition, a temperature-jump experiment on the same system is discussed.

We have also prepared a partially reduced, cyanide inhibited form of the enzyme in which cytochrome a, copper A and copper B are partially reduced and in redox equilibrium. Warming these samples produced absorbance changes at 605 nm which indicate that cytochrome a was becoming more oxidized, but there were no parallel changes in absorbance at 830 nm as would be expected if copper A was becoming reduced. We concluded that electrons were being redistributed from cytochrome a to copper B. The kinetics of the absorbance changes at 605 nm were investigated by temperature-jump methods. Although a rate could not be resolved, we concluded that the process must occur with an (apparent) rate larger than 10,000 s^(-1).

During the course of the temperature-jump experiments, we also found that non-redox related, temperature dependent absorbance changes in fully reduced CO inhibited cytochrome c oxidase, and in the cyanide mixed valence enzyme, took place with an (apparent) rate faster that 30,000 s^(-1).

Reactions of platinum(II) complexes with dioxygen: progress toward alkane functionalization

Whereas stoichiometric activation of C-H bonds by complexes of transition metals is becoming increasingly common, selective functionalization of alkanes remains a formidable challenge in organometallic chemistry. The recent advances in catalytic alkane functionalization by transition-metal complexes are summarized in Chapter I.

The studies of the displacement of pentafluoropyridine in [(tmeda)Pt(CH_3)(NC_5F_5)][BAr^f_4] (1) with γ- tetrafluoropicoline, a very poor nucleophile, are reported in Chapter II. The ligand substitution occurs by a dissociative interchange mechanism. This result implies that dissociative loss of pentafluoropyridine is the rate-limiting step in the C-H activation reactions of 1.

Oxidation of dimethylplatinum(II) complexes (N-N)Pt(CH_3)_2 (N-N = tmeda(1), α-diimines) by dioxygen is described in Chapter III. Mechanistic studies suggest a two-step mechanism. First, a hydroperoxoplatinum(IV) complex is formed in a reaction between (N-N)Pt(CH_3)_2 and dioxygen. Next, the hydroperoxy complex reacts with a second equivalent of (N-N)Pt(CH_3)_2 to afford the final product, (N-N)Pt(OH)(OCH_3)(CH_3)_2. The hydroperoxy intermediate, (tmeda)Pt(OOH)(OCH_3)(CH_3)_2 (2), was isolated and characterized. The reactivity of 2 with several dime thylplatinum(II) complexes is reported.

The studies described in Chapter IV are directed toward the development of a platinum(II)-catalyzed oxidative alkane dehydrogenation. Stoichiometric conversion of alkanes (cyclohexane, ethane) to olefins (cyclohexene, ethylene) is achieved by C-H activation with [(N-N)Pt(CH_3)(CF_3CH_2OH)]BF_4 (1, N-N is N,N'-bis(3,5-di-t- butylphenyl)-1,4-diazabutadiene) which results in the formation of olefin hydride complexes. The first step in the C-H activation reaction is formation of a platinum(II) alkyl which undergoes β-hydrogen elimination to afford the olefin hydride complex. The cationic ethylplatinum(II) intermediate can be generated in situ by treating diethylplatinum(II) compounds with acids. Treatment of (phen)PtEt_2 with [H(OEt_2)_2]Bar^f_4 at low temperatures resulted in the formation of a mixture of [(phen)PtEt(OEt_2)]Bar^f_4 (8) and [(phen)Pt(C_2H_4)H] Bar^f_4 (7). The cationic olefin complexes are unreactive toward dioxygen or hydrogen peroxide. Since the success of the overall catalytic cycle depends on our ability to oxidize the olefin hydride complexes, a series of neutral olefin complexes of platinum(II) with monoanionic ligands (derivatives of pyrrole-2-carboxyaldehyde N-aryl imines) was prepared. Unfortunately, these are also stable to oxidation.

Ancillary ligand effects : from zirconium(IV)-catalyzed homogeneous propylene polymerization to platinum(II)-mediated C-H bond activation

A series of C_s- and C₁-symmetric doubly-linked ansa-metallocenes of the general formula {1,1'-SiMe₂-2,2'-E-('ƞ⁵-C₅H₂-4-R¹)-(ƞ⁵-C₅H-3',5'-(CHMe₂)₂)}ZrC₂ (E = SiMe₂ (1), SiPh₂ (2), SiMe₂ -SiMe₂ (3); R¹ = H, CHMe₂, C₅H₉, C₆H₁₁, C₆H₅) has been prepared. When activated by methylaluminoxane, these are active propylene polymerization catalysts. 1 and 2 produce syndiotactic polypropylenes, and 3 produces isotactic polypropylenes. Site epimerization is the major pathway for stereoerror formation for 1 and 2. In addition, the polymer chain has slightly stronger steric interaction with the diphenylsilylene linker than with the dimethylsilylene linker. This results in more frequent site epimerization and reduced syndiospecificity for 2 compared to 1.

C₁-Symmetric ansa-zirconocenes [1,1 '-SiMe₂-(C₅H₄)-(3-R-C₅H₃)]ZrCl₂ (4), [1,1 '-SiMe₂-(C₅H₄)-(2,4-R₂-C₅H₂)]ZrCl₂ (5) and [1,1 '-SiMe₂-2,2 '-(SiMe₂-SiMe₂)-(C₅H₃)-( 4-R-C₅H₂)]ZrCl₂ (6) have been prepared to probe the origin of isospecificity in 3. While 4 and 3 produce polymers with similar isospecificity, 5 and 6 give mostly hemi-isotactic-like polymers. It is proposed that the facile site epimerization via an associative pathway allows rapid equilibration of the polymer chain between the isospecific and aspecific insertion sites. This results in more frequent insertion from the isospecific site, which has a lower kinetic barrier for chain propagation. On the other hand, site epimerization for 5 and 6 is slow. This leads to mostly alternating insertion from the isospecific and aspecific sites, and consequently, a hemi-isotactic-like polymers. In comparison, site epimerization is even slower for 3, but enchainment from the aspecific site has an extremely high kinetic barrier for monomer coordination. Therefore, enchainment occurs preferentially from the isospecific site to produce isotactic polymers.

A series of cationic complexes [(ArN=CR-CR=NAr)PtMe(L)]⁺[BF₄]⁺ (Ar = aryl; R = H, CH₃; L = water, trifluoroethanol) has been prepared. They react smoothly with benzene at approximately room temperature in trifluoroethanol solvent to yield methane and the corresponding phenyl Pt(II) cations, via Pt(IV)-methyl-phenyl-hydride intermediates. The reaction products of methyl-substituted benzenes suggest an inherent reactivity preference for aromatic over benzylic C-H bond activation, which can however be overridden by steric effects. For the reaction of benzene with cationic Pt(II) complexes, in which the diimine ligands bear 3,5-disubstituted aryl groups at the nitrogen atoms, the rate-determining step is C-H bond activation. For the more sterically crowded analogs with 2,6-dimethyl-substituted aryl groups, benzene coordination becomes rate-determining. The more electron-rich the ligand, as reflected by the CO stretching frequency in the IR spectrum of the corresponding cationic carbonyl complex, the faster the rate of C-H bond activation. This finding, however, does not reflect the actual C-H bond activation process, but rather reflects only the relative ease of solvent molecules displacing water molecules to initiate the reaction. That is, the change in rates is mostly due to a ground state effect. Several lines of evidence suggest that associative substitution pathways operate to get the hydrocarbon substrate into, and out of, the coordination sphere; i.e., that benzene substitution proceeds by a solvent- (TFE-) assisted associative pathway.

Electron flow through cytochrome P450

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).

Part I. Efforts directed towards the total synthesis of shionone. Part II. Conversion of estrone to androst-4-EN-3-ONE: a new method for activating the C-9 and C-10 positions of estrogenic steroids for substitution

Part I: An approach to the total synthesis of the triterpene shionone is described, which proceeds through the tetracyclic ketone i. The shionone side chain has been attached to this key intermediate in 5 steps, affording the olefin 2 in 29% yield. A method for the stereo-specific introduction of the angular methyl group at C-5 of shionone has been developed on a model system. The attempted utilization of this method to convert olefin 2 into shionone is described.

Part II: A method has been developed for activating the C-9 and C-10 positions of estrogenic steroids for substitution. Estrone has been converted to 4β,5β-epoxy-10β-hydroxyestr-3-one; cleavage of this epoxyketone using an Eschenmoser procedure, and subsequent modification of the product afforded 4-seco-9-estren-3,5-dione 3-ethylene acetal. This versatile intermediate, suitable for substitution at the 9 and/or 10 position, was converted to androst-4-ene-3-one by known procedures.

Part I. Kinetics of coarsening of the gamma-prime precipitate in nickel-silicon alloys. Part II. Rate of crystallization of an amorphous Fe-P-C alloy

The coarsening kinetics of Ni₃ Si(γ') precipitate in a binary Ni-Si alloy containing 6.5 wt. % silicon was studied by magnetic techniques and transmission electronmicroscopy. A calibration curve was established to determine the concentration of silicon in the matrix. The variation of the Si content of the Ni-rich matrix as a function of time follows Lifshitz and Wagner theory for diffusion controlled coarsening phenomena. The estimated values of equilibrium solubility of silicon in the matrix represent the true coherent equilibrium solubilities.

The experimental particle-size distributions and average particle size were determined from dark field electron micrographs. The average particle size varies linearly with t^-1/3 as suggested by Lifshitz and Wagner. The experimental distributions of particle sizes differ slightly from the theoretical curve at the early stages of aging, but the agreement is satisfactory at the later stages. The values of diffusion coefficient of silicon, interfacial free energy and activation energy were calculated from the results of coarsening kinetics. The experimental value of effective diffusion coefficient is in satisfactory agreement with the value predicted by the application of irreversible the rmodynamics to the process of volume constrained growth of coherent precipitate during coarsening. The coherent γ' particles in Ni-Sialloy unlike those in Ni-Al and Ni-Ti seem to lose coherency at high temperature. A mechanism for the formation of semi-coherent precipitate is suggested.

Rate and Microstructure Effects on the Dynamics of Carbon Nanotube Foams

Soft hierarchical materials often present unique functional properties that are sensitive to the geometry and organization of their micro- and nano-structural features across different lengthscales. Carbon Nanotube (CNT) foams are hierarchical materials with fibrous morphology that are known for their remarkable physical, chemical and electrical properties. Their complex microstructure has led them to exhibit intriguing mechanical responses at different length-scales and in different loading regimes. Even though these materials have been studied for mechanical behavior over the past few years, their response at high-rate finite deformations and the influence of their microstructure on bulk mechanical behavior and energy dissipative characteristics remain elusive.

In this dissertation, we study the response of aligned CNT foams at the high strain-rate regime of 10² - 10⁴ s^-1. We investigate their bulk dynamic response and the fundamental deformation mechanisms at different lengthscales, and correlate them to the microstructural characteristics of the foams. We develop an experimental platform, with which to study the mechanics of CNT foams in high-rate deformations, that includes direct measurements of the strain and transmitted forces, and allows for a full field visualization of the sample’s deformation through high-speed microscopy.

We synthesize various CNT foams (e.g., vertically aligned CNT (VACNT) foams, helical CNT foams, micro-architectured VACNT foams and VACNT foams with microscale heterogeneities) and show that the bulk functional properties of these materials are highly tunable either by tailoring their microstructure during synthesis or by designing micro-architectures that exploit the principles of structural mechanics. We also develop numerical models to describe the bulk dynamic response using multiscale mass-spring models and identify the mechanical properties at length scales that are smaller than the sample height.

The ability to control the geometry of microstructural features, and their local interactions, allows the creation of novel hierarchical materials with desired functional properties. The fundamental understanding provided by this work on the key structure-function relations that govern the bulk response of CNT foams can be extended to other fibrous, soft and hierarchical materials. The findings can be used to design materials with tailored properties for different engineering applications, like vibration damping, impact mitigation and packaging.

Electron transfer in chemically and genetically modified myoglobins

The temperature dependences of the reduction potentials (E^o') of wildtype human myoglobin (Mb) and three site-directed mutants have been measured by using thin-layer spectroelectrochemistry. Residue Val68, which is in van der Waals contact with the heme in Mb, has been replaced by Glu, Asp, and Asn. At pH 7.0, reduction of the heme iron (III) in the former two proteins is accompanied by uptake of a proton by the protein. The changes in E^o', and the standard entropy (ΔS^o') and enthalpy (ΔH^o') of reduction in the mutant proteins were determined relative to values for wild-type; the change in E^o' at 25°C was about -200 millivolts for the Glu and Asp mutants, and about -80 millivolts for the Asn mutant. Reduction of Fe(III) to Fe(II) in the Glu and Asp mutants is accompanied by uptake of a proton. These studies demonstrate that Mb can tolerate substitution of a buried hydrophobic group by potentially charged and polar residues, and that such amino acid replacements can lead to substantial changes in the redox thermodynamics of the protein.

Through analysis of the temperature dependence and shapes of NMR dispersion signals, it is determined that a water molecule is bound to the sixth coordination site of the ferric heme in the Val68Asp and in the Val68Asn recombinant proteins while the carboxyl group of the sidechain of Glu68 occupies this position in Val68Glu. The relative rhombic distortions in the ESR spectra of these mutant proteins combined with H₂¹⁷O and spin interconversion experiments performed on them confirm the conclusions of the NMRD study.

The rates of intramolecular electron transfer (ET) of (NH₃)₅Ru-His48 (Val68Asp, His81GIn, Cys110AIa)Mb and (NH₃)₅Ru-His48 (Val68GIu,His81GIn,Cys110Ala)Mb were measured to be .85(3)s^-1 and .30(2)s^-1, respectively. This data supports the hypothesis that entropy of 111 reduction and reorganization energy of ET are inversely related. The rates of forward and reverse ET for (NH₃)₅ Ru-His48 (Val68GIu, His81 GIn, Cys110AIa)ZnMb -7.2(5)•10⁴s^-1and 1.4(2)•10⁵s^-1, respectively- demonstrate that the placement of a highly polar residue nearby does not significantly change the reorganization energy of the photoactive Zn porphyrin.

The distal histidine imidazoles of (NH₃)₄isnRu-His48 SWMb and (NH₃)₅Ru-His48 SWMb were cyanated with BrCN. The intramolecular ET rates of these BrCN-modified Mb derivatives are 5.5(6)s^-1 and 3.2(5)s^-1, respectively. These respective rates are 20 and 10 times faster than those of their noncyanated counterparts after the differences in ET rate from driving force are scaled according to the Marcus equation. This increase in ET rate of the cyanated Mb derivatives is attributed to lower reorganization energy since the cyanated Mb heme is pentacoordinate in both oxidation states; whereas, the native Mb heme loses a water molecule upon reduction so that it changes from six to five coordinate. The reorganization energy from Fe-OH₂ dissociation is estimated to be .2eV. This conclusion is used to reconcile data from previous experiments in our lab. ET in photoactive porphyrin-substituted myoglobins proceed faster than predicted by Marcus Theory when it is assumed that the only difference in ET parameters between photoactive porphyrins and native heme systems is driving force. However, the data can be consistently fit to Marcus Theory if one corrects for the smaller reorganization in the photoactive porphyrin systems since they do not undergo a coordination change upon ET.

Finally, the intramolecular ET rate of (NH₃)₄isnRu-His48 SWMb was measured to be 3.0(4)s^-1. This rate is within experimental error of that for (NH₃)₄pyrRu-His48 SWMb even though the former has 80mV more driving force. One likely possibility for this observation is that the tetraamminepyridineruthenium group undergoes less reorganization upon ET than the tetraammineisonicotinamideruthenium group. Moreover, analysis of the (NH₃)₄isnRu-His48 SWMb experimental system gives a likely explanation of why ET was not observed previously in (NH₃)₄isnRu-Cytochrome C.

Synthesis, Oxidation and Photophysics of Perfluoroborated Tetrakis(pyrophosphito)diplatinate (II) and Density Functional Theory (DFT) Study of Electrochemical CO2 Reduction by Mn Catalysts

In the first part of this thesis (Chapters I and II), the synthesis, characterization, reactivity and photophysics of per(difluoroborated) tetrakis(pyrophosphito)diplatinate(II) (Pt(POPBF₂)) are discussed. Pt(POP-BF₂) was obtained by reaction of [Pt₂(POP)₄]^4- with neat boron trifluoride diethyl etherate (BF₃·Et₂O). While Pt(POP-BF₂) and [Pt₂(POP)₄]^4- have similar structures and absorption spectra, they differ in significant ways. Firstly, as discussed in Chapter I, the former is less susceptible to oxidation, as evidenced by the reversibility of its oxidation by I2. Secondly, while the first excited triplet states (T₁) of both Pt(POP-BF₂) and [Pt₂(POP)₄]^4- exhibit long lifetimes (ca. 0.01 ms at room temperature) and substantial zero-field splitting (40 cm^-1), Pt(POP-BF₂) also has a remarkably long-lived (1.6 ns at room temperature) singlet excited state (S₁), indicating slow intersystem crossing (ISC). Fluorescence lifetime and quantum yield (QY) of Pt(POP-BF₂) were measured over a range of temperatures, providing insight into the slow ISC process. The remarkable spectroscopic and photophysical properties of Pt(POP-BF₂), both in solution and as a microcrystalline powder, form the theme of Chapter II.

In the second part of the thesis (Chapters III and IV), the electrochemical reduction of CO₂ to CO by [(L)Mn(CO)₃]^- catalysts is investigated using density functional theory (DFT). As discussed in Chapter III, the turnover frequency (TOF)-limiting step is the dehydroxylation of [(bpy)Mn(CO)₃(CO₂H)]^0/- (bpy = bipyridine) by trifluoroethanol (TFEH) to form [(bpy)Mn(CO)₄]^+/0. Because the dehydroxylation of [(bpy)Mn(CO)₃(CO₂H)]^- is faster, maximum TOF (TOF_max) is achieved at potentials sufficient to completely reduce [(bpy)Mn(CO)₃(CO₂H)]⁰ to [(bpy)Mn(CO)₃(CO₂H)]^-. Substitution of bipyridine with bipyrimidine reduces the overpotential needed, but at the expense of TOF_max. In Chapter IV, the decoration of the bipyrimidine ligand with a pendant alcohol is discussed as a strategy to increase CO₂ reduction activity. Our calculations predict that the pendant alcohol acts in concert with an external TFEH molecule, the latter acidifying the former, resulting in a ~ 80,000-fold improvement in the rate of TOF-limiting dehydroxylation of [(L)Mn(CO)₃(CO₂H)]^-.

An interesting strategy for the co-upgrading of light olefins and alkanes into heavier alkanes is the subject of Appendix B. The proposed scheme involves dimerization of the light olefin, operating in tandem with transfer hydrogenation between the olefin dimer and the light alkane. The work presented therein involved a Ta olefin dimerization catalyst and a silica-supported Ir transfer hydrogenation catalyst. Olefin dimer was formed under reaction conditions; however, this did not undergo transfer hydrogenation with the light alkane. A significant challenge is that the Ta catalyst selectively produces highly branched dimers, which are unable to undergo transfer hydrogenation.

I. The rate and mechanism of the vapor-phase reaction between water and parts-per-million concentrations of nitrogen dioxide. II. The rate and mechanism of the air oxidation of parts-per-million concentrations of nitric oxide in the presence of water vapor.

Part I

A study of the thermal reaction of water vapor and parts-per-million concentrations of nitrogen dioxide was carried out at ambient temperature and at atmospheric pressure. Nitric oxide and nitric acid vapor were the principal products. The initial rate of disappearance of nitrogen dioxide was first order with respect to water vapor and second order with respect to nitrogen dioxide. An initial third-order rate constant of 5.5 (± 0.29) x 10⁴ liter² mole^-2 sec^-1 was found at 25˚C. The rate of reaction decreased with increasing temperature. In the temperature range of 25˚C to 50˚C, an activation energy of -978 (± 20) calories was found.

The reaction did not go to completion. From measurements as the reaction approached equilibrium, the free energy of nitric acid vapor was calculated. This value was -18.58 (± 0.04) kilocalories at 25˚C.

The initial rate of reaction was unaffected by the presence of oxygen and was retarded by the presence of nitric oxide. There were no appreciable effects due to the surface of the reactor. Nitric oxide and nitrogen dioxide were monitored by gas chromatography during the reaction.

Part II

The air oxidation of nitric oxide, and the oxidation of nitric oxide in the presence of water vapor, were studied in a glass reactor at ambient temperatures and at atmospheric pressure. The concentration of nitric oxide was less than 100 parts-per-million. The concentration of nitrogen dioxide was monitored by gas chromatography during the reaction.

For the dry oxidation, the third-order rate constant was 1.46 (± 0.03) x 10⁴ liter² mole^-2 sec^-1 at 25˚C. The activation energy, obtained from measurements between 25˚C and 50˚C, was -1.197 (±0.02) kilocalories.

The presence of water vapor during the oxidation caused the formation of nitrous acid vapor when nitric oxide, nitrogen dioxide and water vapor combined. By measuring the difference between the concentrations of nitrogen dioxide during the wet and dry oxidations, the rate of formation of nitrous acid vapor was found. The third-order rate constant for the formation of nitrous acid vapor was equal to 1.5 (± 0.5) x 10⁵ liter² mole^-2 sec^-1 at 40˚C. The reaction rate did not change measurably when the temperature was increased to 50˚C. The formation of nitric acid vapor was prevented by keeping the concentration of nitrogen dioxide low.

Surface effects were appreciable for the wet tests. Below 35˚C, the rate of appearance of nitrogen dioxide increased with increasing surface. Above 40˚C, the effect of surface was small.

Mechanistic studies of a model reaction for enzymatic hydroxylation

A study has been made of the reaction mechanism of a model system for enzymatic hydroxylation. The results of a kinetic study of the hydroxylation of 2-hydroxyazobenzene derivatives by cupric ion and hydrogen peroxide are presented. An investigation of kinetic orders indicates that hydroxylation proceeds by way of a coordinated intermediate complex consisting of cupric ion and the mono anions of 2-hydroxyazobenzene and hydrogen peroxide. Studies with deuterated substrate showed the absence of a primary kinetic isotope effect and no evidence of an NIH shift. The effect of substituents on the formation of intermediate complexes and the overall rate of hydroxylation was studied quantitatively in aqueous solution. The combined results indicate that the hydroxylation step is only slightly influenced by ring substitution. The substituent effect is interpreted in terms of reaction by a radical path or a concerted mechanism in which the formation of ionic intermediates is avoided. The reaction mechanism is discussed as a model for enzymatic hydroxylation.

The rate and mechanism of the partial oxidation of acetaldehyde by PPM concentrations of nitrogen dioxide

The thermal reaction between nitrogen dioxide and acetaldehyde in the gas phase was investigated at room temperature and atmospheric pressure. The initial rate of disappearance of nitrogen dioxide was 1.00 ± 0.03 order with respect to nitrogen dioxide and 1.00 ± 0.07 order with respect to acetaldehyde. An initial second order rate constant of (8.596 ± 0.189) x 10^-3 1.mole^-1 sec^-1 was obtained at 22.0 ± 0.1 °C and a total pressure of one atmosphere. The activation energy of the reaction was 12,900 cal/mole in the temperature range between 22°C and 122°C.

The products of the reaction were nitric oxide, carbon dioxide, methyl nitrite, nitromethane and a trace amount of trans-dimeric nitrosomethane. The addition of nitric oxide increased the rate of formation of nitromethane and decreased the rate of formation of methyl nitrite. There were no measurable surface effects due to the addition of glass wool or glass beads to the reactor.

Reactants and products were analyzed by gas chromatography. A mechanism was proposed incorporating the principal features of the reaction.