PROJECT SUMMARY:

 The objectives of this research involve the characterization and applications of heme proteins, heme protein microspheres, and synthetic analogs of heme proteins. The reactions of interest include (1) ligand binding to heme proteins and metalloporphyrins, (2) oxygen activation and hydrocarbon oxidation by metalloporphyrins, and (3) the interactions between porphyrins as redox partners in organized media. Some of these studies deal with our recent discovery of a simple sonochemical synthesis of hemoglobin (Hb) and other protein microspheres. Continuation of our efforts in this area, including animal testing, should lead to the development of a new class of blood substitutes based on protein microspheres as O₂ carriers. Other parts of this research provide a fundamental understanding of the molecular mechanisms of heme protein reactions in closely related model metalloporphyrins and oligopeptide-heme complexes.

Transmission electron micrograph of Hb microsphere.

 This should lead (1) to a quantitative understanding of the influences which modulate ligand binding in protein environments, (2) to further characterization and isolation of high oxidation state heme protein intermediates, (3) to a closer understanding of substrate selectivity and regiospecificity by monooxygenases, (4) to fundamental information about interactions in p -overlapping systems such as the photosynthetic reaction center, and (5) to basic knowledge of redox processes in heme proteins.

 A significant portion of our specific aims involve a new and exciting project on proteinaceous microspheres formed during ultrasonic irradiation of hemoglobin (Hb), other heme proteins, and serum albumins. This effort provides an important bridge between our fundamental work on the chemistry of heme proteins and metalloporphyrins and their potential biomedical applications. Some of these applications include biocompatible blood substitutes, magnetic resonance imaging and echocardiographic contrast agents, and novel drug delivery systems. This work is an unexpected cross-fertilization within our group of two diverse and previously completely unrelated projects. It draws on a unique set of research expertise available to no other group in the world: a combination of bioinorganic chemistry with sonochemistry. Ultrasonic irradiation of various proteins (e.g., serum albumin and Hb) creates micron-sized spheres that can be either gas-filled or non-aqueous liquid-filled. We have had substantial success in recent development of proteinaceous microspheres as blood substitutes for O₂ transport, as contrast agents for magnetic resonance imaging, and as spin-label probes for in vivo O₂ and temperature profiling. Microspheres made of Hb and of other protein are currently under development and animal testing as blood substitutes.

 Other work on ligand binding involves the general goal of determining the molecular mechanisms which modulate ligand affinities of metalloporphyrin complexes. In heme proteins and synthetic analogs, O₂ affinities, for example, vary over nearly a million-fold range. The mechanisms by which ligand discrimination is made are critically important to understanding the structure and function of heme proteins. Although many possible mechanisms have been proposed, the ways in which polypeptide-heme interactions affect the reactivity of metalloporphyrins remains largely unexplored. One of our efforts deals with the synthesis of oligopeptide-heme complexes as totally synthetic heme proteins. Here we are learning the origin of heme-protein interactions at its most fundamental level. Specifically, we will continue to examine ligand-binding and redox properties of metalloporphyrin complexes of oligopeptides (15 to 40-mers) in order to assess the nature of these interactions.

 For O₂ activation and high oxidation state chemistry, there are two specific goals: to study shape and polarity selective catalysis of hydrocarbon oxidation by metalloporphyrin complexes and heme proteins and to explicate the nature of the oxidizing intermediates in cytochrome P450. Our first goal is to exemplify various means of molecular recognition (e.g., size, shape, polarity, charge) by use of specifically designed synthetic analogs, including a new class of catalysts: dendrimer-porphyrins. Other proposed work includes further studies of molecular recognition and substrate specificity on the basis of shape, polarity, charge and hydrogen bonding. In work with synthetic metalloporphyrins and dendrimer-porphyrins, we are developing superstructured macrocycles as shape, size, and polarity selective oxidation catalysts for both hydroxylation and epoxidation. As part of this work, we have also developed a new class of nanoporous solids in which highly functionalized porphyrins serve as the building blocks for ionic or hydrogen bonding networks. These solids will be explored as heterogeneous shape-selective catalysts.

Molecular packing diagram of H2(3,5-OHPh)P6 EtOAc showoing the van der Waals surface at 0.7 of atomic radii. Channels of 6.5 x 6.5 Å run along the columns. Solvate molecules are not shown for clarity.

 Our second goal in this area involves the use of heme protein photochemistry to provide a new route to the high oxidation state intermediates in the proteins themselves. Our exploration of the photochemistry of metalloporphyrins has created a new method for the formation of metal-oxo complexes and other high energy species; as part of this, we have attached photo-redox agents have been attached to cytochrome P450 with the intent of the time resolved observation of intermediates.

The active site of cytochrome P450.

 Our interest in the nature of porphyrin-porphyrin interactions is relevant to questions of biological redox processes, especially in the reaction center of photosynthesis with its special pair of chlorophylls and its multiple chlorophyll assemblies. Our goals here involve synthesis and characterization of photophysical properties of two classes of materials: First, bis-porphyrin complexes where large metal ions hold the porphyrins in very close proximity as synthetic analogs of the special pair. The second class are solid state metalloporphyrin assemblies where interactions originate from bridging ligands or from an organizing medium; these are relevant to (but not direct mimics of) electron or energy transport in multi-heme or -chlorophyll proteins (e.g., light harvesting proteins).

A model of the Photosynthetic Reaction Center.
The X-ray crystal structure of Zr(TTP)(2-NHCO-AQ-TTP),
the first bis-porphyrin complex with attached quinone.

 Continuation of our efforts in these areas should lead (1) to the development of a new class of blood substitutes based on protein microspheres as O₂ carriers, (2) to a quantitative understanding of the influences which modulate ligand binding in protein environments, (3) to further characterization and isolation of high oxidation state heme protein intermediates, (4) to a closer understanding of substrate selectivity and regiospecificity by monooxygenases, and (5) to basic knowledge about porphyrin-porphyrin interactions in p -overlapping systems such as the photosynthetic reaction center.

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