To obtain mechanistic insights into the inherent reactivity patterns for copper(I)–O2 adducts, a new cupric–superoxo complex [(DMM-tmpa)CuII(O2•–)]+ (2) [DMM-tmpa = tris((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)amine] has been synthesized and studied in phenol oxidation–oxygenation reactions. Compound 2 is characterized by UV–vis, resonance Raman, and EPR spectroscopies. Its reactions with a series of para-substituted 2,6-di-tert-butylphenols (p-X-DTBPs) afford 2,6-di-tert-butyl-1,4-benzoquinone (DTBQ) in up to 50% yields. Significant deuterium kinetic isotope effects and a positive correlation of second-order rate constants (k2) compared to rate constants for p-X-DTBPs plus cumylperoxyl radical reactions indicate a mechanism that involves rate-limiting hydrogen atom transfer (HAT). A weak correlation of (kBT/e) ln k2 versus Eox of p-X-DTBP indicates that the HAT reactions proceed via a partial transfer of charge rather than a complete transfer of charge in the electron transfer/proton transfer pathway. Product analyses, 18O-labeling experiments, and separate reactivity employing the 2,4,6-tri-tert-butylphenoxyl radical provide further mechanistic insights. After initial HAT, a second molar equiv of 2 couples to the phenoxyl radical initially formed, giving a CuII–OO–(ArO′) intermediate, which proceeds in the case of p-OR-DTBP substrates via a two-electron oxidation reaction involving hydrolysis steps which liberate H2O2 and the corresponding alcohol. By contrast, four-electron oxygenation (O–O cleavage) mainly occurs for p-R-DTBP which gives 18O-labeled DTBQ and elimination of the R group.

In order to characterize the structural and dynamic factors that determine the assembly in b hemoproteins, the solution structure of the 98-residue protein apocytochrome b5 was determined by NMR methods. Over 800 experimental restraints derived from a series of two- and three-dimensional experiments were used. Holocytochrome b5, the protein with iron protoporphyrin-IX liganded to His-39 and His-63, contains in sequence the following elements of secondary structure: beta 1-alpha 1-beta 4-beta 3-alpha 2-alpha 3-beta 5-alpha 4-alpha 5-beta 2-alpha 6 [Mathews, F.S., Czerwinski, E. W., & Argos, P. (1979) The Porphyrins, Vol. 7, pp. 107-147, Academic Press, New York]. The folded holoprotein possesses two hydrophobic cores: an extensive, functional core around the heme (core 1), and a smaller, structural core remote from the heme (core 2). The apoprotein was found to contain a stable four-stranded beta-sheet encompassing beta 1, beta 2, beta 3, and beta 4 and three alpha-helices, corresponding to alpha 1, alpha 2, and alpha 6. Two short alpha-helices (alpha 3 and alpha 5) appear to form partially, and alpha 4 is not detected. These three helices and beta 5 border the heme binding pocket and are disordered in the apoprotein NMR structure. According to backbone 1H-15N NOE results, the most flexible region of the apoprotein, except for the termini, extends from Ala-50 (in beta 5) to Glu-69 (in alpha 5). The polypeptide segment bearing His-63 (located immediately prior to alpha 5) exhibits faster internal motions than that bearing His-39 (at the C-terminal end of alpha 2). The latter imidazole samples a restricted region of space, whereas the former can adopt many orientations with respect to the stable core. It was concluded that heme removal affects the structure and dynamics of most of core 1 whereas it leaves core 2 largely intact. The results provide guidelines for the rational design of b hemoproteins: a modular structure including a packed, stable core and a partially folded binding site is anticipated to present strong kinetic and thermodynamic advantages compared to approaches relying on the complete formation of secondary structure prior to heme binding.

The protonation-reduction of a dioxygen adduct with [LCuI][B(C6F5)4], cupric superoxo complex [LCuII(O2•−)]+ (1), (L=TMG3tren(1,1,1-tris[2-[N2-(1,1,3,3-tetramethylguanidino)]ethyl]amine)), has been investigated. Trifluoroacetic acid (HOAcF) reversibly associates with the superoxo ligand in ([LCuII(O2•−)]+) in a 1:1 adduct [LCuII(O2•−)(HOAcF)]+ (2), as characterized by UV-visible, resonance Raman (rR), nuclear magnetic resonance (NMR) and X-ray absorption (XAS) spectroscopies, along with density functional theory (DFT) calculations. Chemical studies reveal that for the binding of HOAcF with 1 to give 2, Keq = 1.2×105 M−1 (−130 °C) and ΔH° = − 6.9(7) kcal/mol, ΔS° = − 26(4) cal/mol•K). Vibrational (rR) data reveal a significant increase (29 cm−1) in νO-O (= 1149 cm−1) compared to that known for [LCuII(O2•−)]+ (1). Along with results obtained from XAS and DFT calculations, hydrogen bonding of HOAcF to a superoxo O-atom in 2 is established. NMR spectroscopy of 2 at −120 °C in 2-methyltetrahydrofuran are also consistent with 1/HOAcF = 1:1 formulation 2 and that this complex possesses a triplet (S = 1) ground state electronic configuration, as previously determined for 1. The pre-equilibrium acid association to 1 is followed by outer-sphere electron-transfer reduction of 2 by decamethylferrocene (Me10Fc) or octamethylferrocene (Me8Fc), leading to the products H2O2, the corresponding ferrocenium salt and [LCuII(OAcF)]+. Second-order rate constants for electron transfer (ket) were determined to be 1365 M−1 s−1 (Me10Fc) and 225 M−1 s−1 (Me8Fc) at −80 °C. The (bio)chemical relevance of the proton-triggered reduction of the metal-bound dioxygen-derived fragment is discussed.

Upon removal of the heme group, the water-soluble fragment of cytochrome b5 adopts a conformation less stable and compact than that of the holoprotein [Huntley, T. E., & Strittmatter, P. (1972) J. Biol. Chem. 247, 4641-4647]. This conformation, imposed by the amino acid sequence alone, has not been described in detail. One- and two-dimensional proton nuclear magnetic resonance spectroscopy techniques were applied to the apoprotein of the soluble fragment of rat liver cytochrome b5 in an effort to characterize the structure of the apoprotein. Nuclear Overhauser spectroscopy revealed a number of short interresidue distances and demonstrated that, in spite of the increased flexibility, at least one cluster of side chains exists on a time scale long enough for study. Several residues participating in the cluster, in particular the only Trp (Trp 22), were identified. Similarities with the spectrum of the reduced holoprotein were observed that led to the inspection of the cytochrome b5 crystal structure for assigning resonances. It appeared that the environment of this residue maintains its integrity in the apoprotein. Since in the holoprotein Trp 22 belongs to a hydrophobic core formed in part by beta-strands, it is proposed that some of this beta-structure is stable in the absence of the heme-protein interactions. Implications for structure and folding are discussed.

Apocytochrome b5 is a partially folded protein which contains a stable structural unit under native conditions [Moore, C.D., Al-Misky, O.N., & Lecomte, J.T.J. (1990) Biochemistry 30, 8357-8365]. In this work, the fold of the unit was examined by using 1H and 15N-edited two-dimensional NMR spectroscopy. It was found that it contains four of the five beta-strands and two of the six alpha-helices present in the holoprotein. The remainder of the structure appears to be mostly unstructured and fluctuating among several conformations. The structural unit is stabilized by a hydrophobic core formed by residues from each of the folded elements of secondary structure. Nuclear Overhauser effects and chemical shift values demonstrated that the unit is structurally similar in the apo- and holoproteins. However, the backbone amide hydrogen exchange was found to be much accelerated in the apoprotein. The paramagnetic relaxation agent HyTEMPO was used to probe the packing of the structure. HyTEMPO has unrestricted access to the empty heme binding site whereas it is unable to penetrate the stabilizing core. It was concluded that addition of the heme is necessary for the last strand to dock properly to the rest of the sheet. The kinetics of refolding of the apoprotein were monitored by stopped-flow fluorescence spectroscopy. Extensive protection of the sole tryptophan residue by docking of the two polypeptide termini occurs in less than 60 ms. It was proposed that apocytochrome b5, with its two-region behavior, might serve as a model for the design of proteins which bind a prosthetic group.

Due to its inherent reactivity, nitroxyl (HNO), must be generated in situ through the use of donor compounds, but very few physiologically useful HNO donors exist. Novel N-substituted hydroxylamines with carbon-based leaving groups have been synthesized, and their structures confirmed by X-ray crystallography. These compounds generate HNO under nonenzymatic, physiological conditions, with the rate and amount of HNO released being dependent mainly on the nature of the leaving group. A barbituric acid and a pyrazolone derivative have been developed as efficient HNO donors with half-lives at pH 7.4, 37 °C of 0.7 and 9.5 min, respectively.

A new tool for analyzing compound libraries by NMR has been developed. Aliquots of solution-state samples (between 120 and 350 microL) are directly injected, using a standard liquids handler, into an NMR (LC-NMR) flow probe. Automated NMR software tracks--and suppresses--intense signals arising from the nondeuterated solvents used (if any) and acquires high-sensitivity one-dimensional 1H NMR spectra. An 88-member combinatorial library, dissolved in DMSO and stored in a 96-well microtiter plate, has been analyzed a number of ways using this technique. This nondestructive technique, which we call direct-injection NMR (DI-NMR) and which is embodied in our versatile automated sample changer (VAST) hardware, has proven to be both routine and robust. Our success in automatically acquiring the NMR data for entire plates of library compounds (within 4-8 h) has caused us to develop new ways to display and analyze the resulting NMR data, as will be shown here.

Current interest in copper/dioxygen reactivity includes the influence of thioether sulfur ligation, as it concerns the formation, structures, and properties of derived copper-dioxygen complexes. Here, we report on the chemistry of {L-CuI}2-(O2) species L = DMMESE, DMMESP, and DMMESDP, which are N3S(thioether)-based ligands varied in the nature of a substituent on the S atom, along with a related N3O(ether) (EOE) ligand. CuI and CuII complexes have been synthesized and crystallographically characterized. Copper(I) complexes are dimeric in the solid state, [{L-CuI}2](B(C6F5)4)2, however are shown by diffusion-ordered NMR spectroscopy to be mononuclear in solution. Copper(II) complexes with a general formulation [L-CuII(X)]n+ {X = ClO4–, n = 1, or X = H2O, n = 2} exhibit distorted square pyramidal coordination geometries and progressively weaker axial thioether ligation across the series. Oxygenation (−130 °C) of {(DMMESE)CuI}+ results in the formation of a trans-μ-1,2-peroxodicopper(II) species [{(DMMESE)CuII}2(μ-1,2-O22–)]2+ (1P). Weakening the Cu–S bond via a change to the thioether donor found in DMMESP leads to the initial formation of [{(DMMESP)CuII}2(μ-1,2-O22–)]2+ (2P) that subsequently isomerizes to a bis-μ-oxodicopper(III) complex, [{(DMMESP)CuIII}2(μ-O2–)2]2+ (2O), with 2P and 2O in equilibrium (Keq = [2O]/[2P] = 2.6 at −130 °C). Formulations for these Cu/O2 adducts were confirmed by resonance Raman (rR) spectroscopy. This solution mixture is sensitive to the addition of methylsulfonate, which shifts the equilibrium toward the bis-μ-oxo isomer. Further weakening of the Cu–S bond in DMMESDP or substitution with an ether donor in DMMEOE leads to only a bis-μ-oxo species (3O and 4O, respectively). Reactivity studies indicate that the bis-μ-oxodicopper(III) species (2O, 3O) and not the trans-peroxo isomers (1P and 2P) are responsible for the observed ligand sulfoxidation. Our findings concerning the existence of the 2P/2O equilibrium contrast with previously established ligand-CuI/O2 reactivity and possible implications are discussed.