EPR Spectroscopic Studies of the Active Sites of Some Heme- and Copper-Containing, Oxygen-Binding Proteins

Citation

Morse, Randall Heywood (1981) EPR Spectroscopic Studies of the Active Sites of Some Heme- and Copper-Containing, Oxygen-Binding Proteins. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/7cva-0k93. https://resolver.caltech.edu/CaltechTHESIS:03132018-112023831

Abstract

All oxygen-utilizing proteins contain copper, iron, or both, at the oxygen-bonding sites. The ligand environment and geometry about the metal centers in these proteins must be crucial in determining their individual functions. This thesis reports studies in which the ligand nitric oxide is used as a spin probe to investigate the structure of the oxygen-binding sites of myoglobin and some inorganic heme analogues, and of tree and fungal laccase and cytochrome c oxidase.

Nitric oxide reacts with heme iron in ferrous hemeproteins such as hemoglobin and myoglobin, to form six-coordinate paramagnetic complexes. Chapter II reports investigations on the EPR spectra of the nitric oxide complexes of ferrous myoglobin, cytochrome c, and Fe(II) protoporphyrin IX-imidazole, which change with temperature over the range 30-180 K. This temperature dependence could be due to motional/relaxation effects or to a chemical equilibrium. To resolve this matter, the technique of factor analysis was used to deconvolute the temperature-dependent EPR spectra. By this method it has been found that all of the spectra for any given complex can be reproduced by adding together varied amounts of two signals, demonstrating that the variation of the EPR spectra with temperature is due to an equilibrium between two species. The two species differ in enthalpy by no more than about 2 kcal/mol. The observed signals are interpreted as arising from two six-coordinate conformers of the nitrosylheme∙nitrogen base complexes, differing primarily in the position of the iron with respect to the ligands and to the heme plane. The anomalous behavior of the terminal respiratory enzyme cytochrome c oxidase, which exhibits only one EPR signal independent of temperature, suggests that the NO-oxygen may interact with the cuprous ion at the cytochrome a₃-Cu_a3 site of the enzyme. These results have implications for studies in general on oxygen-carrying proteins.

In addition to being a strong-field ligand, NO is also a reactive molecule which can be oxidized to NO^-₂ and NO^-₃, and reduced to N₂O, N₂ and NH₃. Chapter III details investigations of the reactions of NO with the copper-containing oxidases tree and fungal laccase, as well as with tree laccase depleted in type 2 copper. The oxidation states of the enzymes were monitored by EPR and optical spectroscopy, and the reaction products of NO were determined by NMR and mass spectroscopy. These studies show that NO reduces all three copper sites of fungal laccase. In addition, NO forms a specific complex with the reduced type 2 copper. NO similarly reduces all of the copper sites in tree laccase, but it also oxidizes the reduced sites produced by ascorbate or NO reduction. A catalytic cycle is set up in which N₂O, NO^-₂ and various forms of the enzyme are produced. On freezing of fully reduced tree laccase in the presence of NO, the type 1 copper becomes reoxidized. This reaction does not occur with the enzyme depleted in type 1 copper, suggesting that it involves intramolecular electron transfer from the type 1 copper to NO bound to the type 2 copper. When the half-oxidized tree laccase is formed in the presence of NO, a population of molecules exists which exhibits a type 3 EPR signal. A triplet EPR signal is also seen in the same preparation, and is attributed to a population of the enzyme molecules in which NO is bound to the reduced copper of a half-oxidized type 3 copper site. The implications of these results towards the structures of tree and fungal laccase are discussed.

Nitric oxide has also been used to probe the structure of the metal centers of cytochrome c oxidase, resulting in the discovery of three stable conformations of the oxidized enzyme [G. W. Brudvig, T. H. Stevens, R. H. Morse and S. I. Chan (1981) Biochemistry 20, in press]. These conformat1onsdiffer in the structure of the cytochrome a₃-Cu_a3 site, which is the site of oxygen reduction, and are distinguishable by EPR spectroscopy. Two of the diagnostic EPR signals are unusual in that they cannot be interpreted simply in terms of normal Cu(II) or Fe(III) EPR signals. The "g5" conformation identified as a transient species occurring upon reoxidation of the reduced enzyme by O₂ [R. W. Shaw, R. E. Hansen and H. Beinert (1978) J. Biol. Chem. 253, 6637-6640] similarly exhibits an EPR signal not subject to simple interpretation. In view of the complexity of these three EPR signals, they are most likely due to the coupled cytochrome a₃⁺³-Cu_a3⁺² site of the oxidized enzyme. Chapter IV describes calculations of the energy levels of, and allowed EPR transitions from, the cytochrome a₃-Cu_a3 site under various conditions of exchange coupling and dipolar coupling, as a function of other parameters such as the magnitude of the rhombic zero-field splitting of the heme iron and distance between the two metal ions. On the basis of these calculations, one of the three unusual EPR signals from oxidized cytochrome c oxidase is deduced to arise from a strongly exchange-coupled (|J| > 200 cm^-1) cytochrome a₃⁺³-Cu_a3⁺² site, one from a weakly exchange-coupled (|J| > 0.25 cm^-1) cytochrome a₃⁺³-Cu_a3⁺² site, and one from an admixture of S = 5/2 and S = 3/2 states of the cytochrome a₃⁺³ site.

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