We studied the selectivity of a functional model of cytochrome c oxidase's active site that mimics the coordination environment and relative locations of Fe(a3), Cu(B), and Tyr(244). To control electron flux, we covalently attached this model and analogs lacking copper and phenol onto self-assembled monolayer-coated gold electrodes. When the electron transfer rate was made rate limiting, both copper and phenol were required to enhance selective reduction of oxygen to water. This finding supports the hypothesis that, during steady-state turnover, the primary role of these redox centers is to rapidly provide all the electrons needed to reduce oxygen by four electrons, thus preventing the release of toxic partially reduced oxygen species.

A functional heme/nonheme nitric oxide reductase (NOR) model is presented. The fully reduced diiron compound reacts with two equivalents of NO leading to the formation of one equivalent of N 2O and the bis-ferric product. NO binds to both heme Fe and nonheme Fe complexes forming individual ferrous nitrosyl species. The mixed-valence species with an oxidized heme and a reduced nonheme Fe B does not show NO reduction activity. These results are consistent with a so-called ''trans'' mechanism for the reduction of NO by bacterial NOR.functional model ͉ NO reduction ͉ N2O ͉ "trans" mechanism N itric oxide reductase (NOR) is a membrane-bound enzyme that catalyzes the 2e Ϫ reduction of nitric oxide (NO) to nitrous oxide (N 2 O), an obligatory step involved in the sequential reduction of nitrate to dinitrogen known as bacterial denitrification. The active site of NOR consists of a monohistidine ligated five-coordinate heme and a trisimidazole ligated nonheme Fe B . This structure strongly resembles the active site of oxygen reduction enzyme-cytochrome c oxidase (CcO), which possesses a heme-a 3 /Cu B center ( Fig. 1) (1, 2). Essentially, the distal metal Cu B in CcO is replaced by a nonheme Fe metal in NOR; NOR and CcO are thought to be distant relatives.The dinuclear iron active site in NOR was confirmed a decade ago by spectroscopic studies (3). Presumably, two NO molecules are turned over to give one molecule of N 2 O and one molecule of H 2 O at the diiron center with the consumption of two electrons and two protons. Although many enzyme studies of NOR have been focused on the intermediate trapping and elucidation of the reaction mechanism (4-15), the details of the catalytic cycle are still unresolved because of the lack of structural information and uncertainty regarding short-lived intermediates.In contrast to enzyme studies, synthetic biomimetic model complexes provide a straightforward and controlled method to understand how this chemical transformation proceeds at the enzyme active site. However, only a few synthetic models have been developed that mimic the active site of NOR; moreover, these compounds either lack a proximal imidazole ligand (16,17) or use pyridine as a replacement for the histidine ligands (18)(19)(20). No functional NOR models have been reported to date. Our CcO model complexes have proved to be functionally active for oxygen reduction reaction with minimal reactive oxygen species (ROS) formation (21-24). These appear to be promising NOR model candidates if the distal Cu metal is replaced by an iron because the resulting diiron compound has almost all of the key components in NOR: a heme Fe with a proximal imidazole ligand and a trisimidazole ligated nonheme Fe center.In this report, we disclose the first synthetic functional NOR model LFe II /Fe II (Fig. 2), which reacts with two equivalents of NO to give one equivalent of N 2 O and the bis-ferric product. We have shown that NO binds to both heme Fe and Fe B to form a possible bis-nitrosyl intermediate; subsequently, the two bound NO molec...

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Five iron porphyrins with different superstructures were immobilized on self-assembled-monolayer (SAM)-coated interdigitatedarray (IDAs) gold-platinum electrodes. The selectivity of the catalysts i.e., limited formation of partially reduced oxygen species (PROS) in the electrocatalytic reduction of dioxygen, is a function of 2 rates: (i) the rate of electron transfer from the electrode to the catalyst, which is controlled by the length, and conjugation of the linker from the catalyst to the electrode and (ii) the rate of bound oxygen (superoxide) hydrolysis, which correlates with the presence of a water cluster in the gas-binding pocket influencing the rate of oxygen binding; these factors are controlled by the nature of the porphyrin superstructure. The structurally biomimetic Trisimidazole model is the most selective.electron transfer rate ͉ oxygen binding rates ͉ water cluster ͉ oxygen complex stabilization ͉ partially reduced oxygen species A recent report showed that water may inhibit oxygen binding in hemoprotein models (1). This echoes the ''water displacement model'' proposed for oxygen binding in myoglobin (2-4). This finding is relevant to cytochrome c oxidase (CcO) for 2 reasons: (i) CcO binds and reduces oxygen to form water (5) and (ii) the hydrolysis of the oxygen complex in CcO and in other enzymes is an issue and occurs when water, protons, and electrons are present (6, 7). This reaction releases partially reduced oxygen species (PROS) that are toxic to organisms (Fig. 1). The study herein examines the formation of PROS during the electrocatalytic reduction of oxygen by several site-isolated CcO models ( Fig. 2) as a function of (i) the oxygen association rate [k on (O 2 )] and the rate of hydrolysis, both associated with the presence of water in the gas binding pocket, and (ii) the availability of electrons at the oxygen reduction site associated with the rate of electron arrival from the electrode to the catalyst [k(eT)] and/or the presence of redox active species in the vicinity of the heme. The latter aspects were specifically addressed in previous studies by immobilizing the active-site models onto self-assembled-monolayer (SAM)-coated rotating ring disk gold electrodes (RRDE) (8-10): k(eT) was controlled by the length and conjugated character of the linker between the catalyst and the gold electrode, and immobilization allowed site isolation. To get better collection efficiencies for the detection of PROS and to prevent damage to the SAM associated with high rotation rates, the present study used Au-Pt interdigitated array electrodes (IDAs). The collection efficiency of these IDA electrodes is in the 65-90% range as compared with Ͻ20% in RRDE (8).IDAs have 2 sets of alternating arrays of electrodes. The electrode widths and spacing are on similar dimensions, and this distance is within diffusion layers for typical electroactive species in solution. This results in diffusion gradients of adjacent microband electrodes that overlap with each other. Redox products created at the Au generator ...

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