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.