Structural Mechanisms of Nonplanar Heroes in Proteins

The objective is to assess the occurrence of nonplanar distortions of hemes and other tetrapyrroles in proteins and to determine the biological function of these distortions. Recently, these distortions were found by us to be conserved among proteins belonging to a functional class. Conservation of the conformation of the heme indicates a possible functional role. Researchers have suggested possible mechanisms by which heme distortions might influence biological properties; however, no heme distortion has yet been shown conclusively to participate in a structural mechanism of hemoprotein function. The specific aims of the proposed work are: (1) to characterize and quantify the distortions of the hemes in all of the more than 300 hemoprotein X-ray crystal structures in terms of displacements along the lowest-frequency normal coordinates, (2) to determine the structural features of the protein component that generate and control these nonplanar distortions by using spectroscopic studies and molecular-mechanics calculations for the native proteins, their mutants and heme-peptide fragments, and model porphyrins, (3) to determine spectroscopic markers for the various types of distortion, and, finally, (4) to discover the functional significance of the nonplanar distortions by correlating function with porphyrin conformation for proteins and model porphyrins. The normal structural decomposition (NSD) method is crucial to successfully achieving these goals. NSD is a new computational procedure for quantifying the distortions of porphyrins, especially in protein crystal structures. Using the NSD procedure, the heme groups in X-ray structures are found to show different conserved distortions for different functional classes of proteins. The NSD method in combination with molecular mechanics and spectroscopic studies provide the means for discovering the structural mechanisms that cause the different types of distortion. Understanding the influence of heme nonplanarity on the function of heme proteins has substantial significance for understanding allosteric control mechanisms in biology and for the purpose of developing biomimes for use in healthrelated and commercial processes. JJ Principal Investigator/Program Director (Last, first, middle): Shelnutt, John A. PHS398 (Rev. 5/95) Page __2__ JJ a. Introduction Our ultimate goal is to determine the functional significance of nonplanar heme distortions in hemoproteins. It has been recognized for about 10 years that the hemes in many hemoproteins are highly distorted from planarity and that these nonplanar distortions might play a role in their biological function. In related photosynthetic proteins, nonplanar distortions have been suggested to influence the redox properties of chlorophyll pigments, with consequent effects on electron-transfer rates in photosynthetic reaction centers and antennae complexes. More recently, by using a new method for characterizing and quantifying these distortions, our group has found that these distortions are often of different types for proteins with different functions, and the types of distortion are conserved for proteins belonging to the same functional class. This suggests even more strongly that the biological function of hemoproteins might be modulated by protein control over the conformation of the heme prosthetic group. The importance of the nonplanar distortions of the heme is also emphasized by recent studies of model nonplanar porphyrins showing, first, that hemes are expected to be nearly planar in absence of the protein moiety and, second, that the nonplanar structure influences relevant chemical and photophysical properties (e.g., axial ligand affinity, redox potentials, transition dipoles and energies). The first specific aim of the proposed work is to evaluate all of the 325 heme protein X-ray crystal structures in the Protein Data Bank using the newly developed normal structural decomposition (NSD) procedure. This will quantify and characterize the heme conformational motifs present in the hemoproteins and the degree to which these motifs are maintained within functional classes of proteins. Already over 100 hemes of the proteins have been characterized with NSD and, in most cases, the nonplanar structures have been found to be characteristic of the specific protein types. The structural decomposition of all of the hemes in the Protein Data Bank will also delineate the effects of natural amino acid sequence variation, mutation, axial ligation, and other protein differences on the conformation of the heme. Another aim is to relate the primary, secondary, and tertiary structure of the protein moiety to the particular conformation of the heme. In the case of the c-type cytochromes, a mechanism for producing the strong ruffling of the heme skeleton is suggested by the NSD results. We expect that NSD characterization of the hemes in other proteins will lead to other hypotheses relating protein structure and heme conformation. Based on these hypotheses, spectroscopic and molecular modeling studies will be carried out to verify or disprove the structural mechanisms proposed. The methods to be used in these studies are illustrated by our preliminary results for nickel-reconstituted cytochrome c and nickel microperoxidase-11, in which a proposed mechanism of proteinheme interaction leading to the ruffling of the heme in the cytochromes c is tested. Studies of model nonplanar porphyrins are also necessary to improve the ability of spectroscopic techniques to distinguish the types and magnitudes of distortion of the heme. Currently, resonance Raman spectroscopy allows one to distinguish the magnitude of nonplanar distortion but not the type (e.g., doming, ruffling, saddling, etc.). In addition, at present it is unknown whether the relationship between Raman line frequencies and the magnitude of distortion varies with the type of distortion. Further improvements in the spectroscopic probes of porphyrin structure are another specific goal of the proposed work. Molecular modeling studies coupled with the spectroscopic data and the NSD results provide critical insight into the possible mechanisms by which the surroundings of the porphyrin can induce various nonplanar distortions. A molecular mechanics force field has been developed and exhaustively validated experimentally for the prediction of porphyrin conformations. The molecular modeling will provide information about the energetics of nonplanar distortions, aiding in the determination of their functional significance and structural origin. JJ Principal Investigator/Program Director (Last, first, middle): Shelnutt, John A. PHS398 (Rev. 5/95) Page __3__ JJ Finally, the relationships between chemical function and heme structure will be directly examined in proteins and model porphyrin systems. For example, a series of porphyrins in which the magnitude of the nonplanarity is varied by changing the bulkiness of the peripheral substituents will be used to investigate the effect of each type of nonplanar distortion on axial ligation and other chemical properties. Such investigations will determine the magnitude of the effects induced by nonplanar distortion and the different ways in which nonplanar conformers can influence chemical properties. In the proteins, NSD results for heme proteins will be correlated to functional properties in as much as is possible. Also, protein function will be varied by mutation and by changing the pH and other solution properties, so that possible correlations with the spectroscopically-determined heme conformations can be ascertained. b. Background and Significance Structure-Function Relationships in Hemoproteins. Since Warburg's work beginning over 70 years ago, the heme proteins have been intensely investigated with the aim of determining the structural mechanisms controlling their varied biological functions. The major remaining questions concern the role of the protein in modulating the properties of the iron-porphyrin cofactor to yield the specific biological functions, e.g., electron transfer (cytochromes), O2 transport and storage (hemoglobin, myoglobin), respiration (cytochrome oxidase), or catalysis (peroxidase, monooxygenase, catalase). The immediate surroundings of the heme active site certainly have a dominant influence on function; in particular, axial coordination to the central iron atom, covalent attachment of the heme to the protein, and amino acid side chains in the immediate vicinity of the active site are all important. However, subtle influences on the structure of the active site are sometimes observed to modify the activity of the protein. For example, in the cytochromes the number, H-bonding interactions, conformations, and nature of the axial ligands appear to be of primary importance in governing the redox properties. As another example, the hemes in hemoglobin have oxygen affinities that depend on whether the other hemes have O2 bound as an axial ligand or not. Heme affinity differences in hemoglobin have been ascribed to subtle structural changes in axial coordination to the iron atom which are transmitted from one heme to the others through the protein's tertiary and quaternary structure. It is also possible that conformational differences in the heme itself may influence the reactivity of heme proteins. To arrive at a detailed structural understanding of the function of heme proteins requires a thorough knowledge of the various influences of structure on function. In many instances, the structure of the heme is ∆ z (Å ) -0.5 0.0 0.5 I II III IV