Stephen Fox

Copper-dioxygen chemistry and modeling the Fe-Cu center in cytochrome c oxidase

The copper(1) complex [(TMPA)Cu(RCN)]+ (1) binds 02 forming [{ (TMPA)Cu}2(02)]2+ (2), with trans-y-1, 2 peroxo-coordination. Ligands with quinolyl groups substituting for the pyridyl donors in TMPA cause dramatic changes in the course of reaction, in one case stabilizing a CdO2 1:l adduct [(BQPA)Cu(02)]+ (6). The kinetics/thermodynamics are compared. Reaction of 1 with (F 8-TPP)Fe(II)pip2 (8) and 02 yields the p-0x0 species [(Fg-TPP)Fem-O-Cun(TMPA)]+ (9); this reversibly protonates giving p-hydroxo bridged [(Fg-TPP)Fe-(OH)-Cu(TMPA)]2+ (12). The novel NMR properties of 9 are described. These complexes are discussed in terms of their model 02-chemistry in hemocyanins or cytochrome c oxidase.

Investigations of dioxygen reactivity with synthetic models of the metalloproteins hemocyanin and cytochrome c oxidase

Synthetic, chemical, and low-temperature stopped-flow kinetic studies in EtCN indicate initial formation of the 1:l 02 adduct [(TMPA)Cu(O2)]+ (4), and subsequent formation of the 2:1 02 adduct [( (TMPA>CU]~(O~)]~ + (2) from [ (TMPA)Cu(RCN)]+ (1) and 02; vacuum-cycling experiments demonstrate reversible binding of a. An X-ray structural study of 2 revealed a trans-p-1, 2 configuration for the 022- ligand; thus, the reactivity of 1 with 02 models the function of hemocyanin. Reaction of 1 with (Fg-TPP)Fe(II)pip2 (5) and 02, yielded the structurally characterized p-0x0 [(Fg-TPP)Fe(III)-0-Cu(II)(TMPA)]+ (6) species, which was protonated to yield the p-hydroxo bridged analogue [(Fg TPP)Fe-(OH)-Cu(TMPA)I2+ (9); these complexes may model intermediates in the OZ reduction cycle, and/or the resting-state, of cytochrome c oxidase.

Dioxygen Reactivity in Copper Proteins and Complexes

Copper ion is an essential trace element found in living systems, and its importance resides in its role as a protein or enzyme active-site constituent. Thus, copper proteins occur widely in nature, performing a diverse array of functions (Adman, 1991; Beinert, 1991; Kitajima, 1992; Solomon et al., 1992; Karlin and Tyeklar, 1993). All of these involve oxidatioN-reduction (i.e., “redox”) activity, since a number of oxidation states are known for copper ion (Hathaway, 1987). Ligand donors for copper which are typically available in protein matrices include the side-chain imidazole group of histidine, the phenol oxygen donor of tyrosine, or the sulfur atom of the thiol group from cysteine. With combinations of these, the CuI and CuII oxidation states are readily accessible and interconvertible under physiological conditions, using available oxidants (e.g., molecular oxygen [dioxygen; O2]) or reducing agents such as glutathione (γ-Glu-Cys-Gly) a sulthydrylcontaining tripeptide which is abundant (~1 mM) in biological media, or ascorbic acid (i.e., vitamin C).

Copper–dioxygen reactivity involved in the formation of µ-oxo [(por)FeIII–O–CuIIL]+ heterodinuclear complexes (por = porphyrinate, L = tetradentate ligand), and novel synthesis of square-planar FeII(por) species

In the reaction of FeIII(por) species with [LCuI(KMeCN)]+ and O2 to give µ-oxo [(por)FeIII–O–CuIIL]+3(por = porphyrinate, L = tetradentate ligand), copper–dioxygen adducts or their decomposition products must be present, otherwise [(por)FeIII–OH] or [(por)FeIII–O–FeIII(por)] products appear; a novel synthesis ot square-planar FeII(por) is also described.

A Chemical Signature for Cytidine Acetylation in RNA

N4-acetylcytidine (ac4C) is a highly conserved modified RNA nucleobase whose formation is catalyzed by the disease-associated N-acetyltransferase 10 (NAT10). Here we report a sensitive chemical method to localize ac4C in RNA. Specifically, we characterize the susceptibility of ac4C to borohydride-based reduction and show this reaction can cause introduction of noncognate base pairs during reverse transcription (RT). Combining borohydride-dependent misincorporation with ac4Cs known base-sensitivity provides a unique chemical signature for this modified nucleobase. We show this unique reactivity can be used to quantitatively analyze cellular RNA acetylation, study adapters responsible for ac4C targeting, and probe the timing of RNA acetylation during ribosome biogenesis. Overall, our studies provide a chemical foundation for defining an expanding landscape of cytidine acetyltransferase activity and its impact on biology and disease.

Dioxygen Reactivity Models for Cytochrome C Oxidase: Synthesis and Characterization of Oxo and Hydroxo-Bridged Porphyrin-Iron/Copper Dinuclear Complexes

The synthesis of appropriate transition-metal complexes to model the structural, spectroscopic, and magnetic properties of a metalloprotein active-site provides an opportunity to consider the function and associated mechanism of that metalloprotein at the molecular level. One nice example is the dinuclear cuprous amine-bis-pyridyl complex, which effects arene hydroxylation (albeit of the ligand m-xylyl spacer) using molecular oxygen (O2).1 This extraordinary reaction involves cleavage of the O-O bond and subsequent insertion of an oxygen atom into an arene C-H bond under essentially ambient conditions, to model the function of copper monooxygenases such as tyrosinase. Another excellent example is the generation of dicupric trans-µ-1,2-peroxo complexes from cuprous precursors and O2, reversibly,2–4 to model the oxygen-transport property of the protein hemocyanin, which subsequently was discovered to bind O2 in η2:η2 fashion, as shown in Figure 1.4 The metalloprotein cytochrome c oxidase,5 however, due to its combination of diverse and unusual active-site metal centers, has eluded a convincing model description. As for its function, it probably binds O2 at a dinuclear site comprising heme-iron and histidyl-copper coordination; it then cleaves the O-O bond, via reduction, (vide infra ).5 The structural changes associated with this dinuclear site during turnover, and the intermediates produced therefrom, are by no means clearly understood. In the resting state, the dinuclear site exhibits strong antiferromagnetic coupling (-J =200 cm-1) suggesting the involvement of a bridging ligand, often postulated as µ-sulfido, µ-chloro, or µ-hydroxo. Thus, we have endeavored to synthesize model complexes of this enigmatic dinuclear site.