Fourier Transform Infrared And Resonance Raman Characterization Of Cytochrome Ba 3 From Thermus Thermophilus

ABSTRACT Resonance Raman and Fourier transform infrared spectra of several derivatives of cytochrome bas, a newly discovered terminal oxidase of the bacterium Thermus thermophilus, are reported. The RR features characteristic of cytochrome 33 are uniquely observed without interference from cytochrome b by subtraction of the analogous cytochrome bs spectra. The spin state indicator peaks of the as heme appear at unusually high frequencies, suggesting a uniquely small heme core size. Multiple C-O and Fe-N(Im) peaks are observed in the FTIR and RR spectra, respectively. Their relative intensities are temperature-dependent suggesting the presence of discrete interconverting conformers of the enzyme. Thermodynamic parameters for interconversion of these conformers are derived. The C-O infrared stretching frequencies of the fully reduced carbon-monoxy enzyme show that CO binds to CUB+ following photodissociation of CO from the heme 33 at all temperatures up to ambient. At temperatures > 200 K, the Cu-bound CO returns to the heme a^ in a first-order thermal back reaction; below 200 K, the CuB+-CO complex is kinetically stable. The activation parameters of this reaction, AH* = 14.9 kcal/mol and AS* = -5 cal/mol/K, were obtained by observing the kinetics of this reaction from 205-230 K and at 300 K. These are compared to analogous parameters obtained previously for mitochondrial cytochrome aa3 preparations (F.G. Fiamingo, R.A. Altschuld, P.P. Moh, and J.O. Alben, J. Biol. Chem. 257, 1639-1650 (1982)). These results demonstrate unusual features of the cytchrome ba3 enzyme and suggest unsuspected mechanistic possibilties in the ligand binding and function of cytochrome oxidases in general.1. INTRODUCTIONThe presence of more than one type of terminal oxidase is quite common among bacteria (c/Ref. 1 for review). A new type of cytochrome oxidase, cytochrome ba3, has recently been purified from the plasma membrane of the bacterium Thermus thermophilus (2). Uniquely for cytochrome oxidase, the enzyme contains spectroscopically distinct hemes, one heme 33 and one b-type heme chromophore. The cytochrome b apparently assumes the functional role of cytochrome a in the aa3 oxidases. The enzyme also contains one CUA center and one CUB-type center, analogous to the copper centers of cytochromes aa3 (2,3). In addition to its own intrinsic interest, cytochrome ba3 promises to be an important model for the aa3-containing oxidases; it offers a unique opportunity for characterization of the optical and redox properties of cytochrome 33 without interferences from a second heme a-type cytochrome (2).The mechanism detailing how dioxygen binds to reduced heme 33 and is reduced to water is poorly understood. Due to great rapidity of the reaction between reduced cytochrome oxidase and dioxygen, attempts to identify oxygen-bound intermediates at room temperature by conventional rapid reaction methods are subject to difficulties and ambiguities. Carbon monoxide, a competitive inhibitor of O2, has been used as an IR-active structural probe for the oxygen binding site of cytochrome oxidase and other O2-binding proteins (4-6). The CO-IR frequency (near 2000 cm'1 ) and bandwidth are highly sensitive to the environment around the CO ligand and can provide valuable information about the nature of the heme a3- copper binding site (6).Under physiological conditions CO binds to the reduced heme 33 of cytochrome oxidase and forms a stable complex. CO can be photodissociated from the heme either at ambient or cryogenic temperature, and the kinetics of its rebinding to the heme can be observed. Previous IR studies have established (7,8) that at low temperature in cytochrome aa3, the photodissociated CO binds quantitatively to the nearby CU(!)B before returning to the 33 heme. This has not, however, been observed at ambient temperature. The question of the transient fate of photodissociated CO is crucial in mechanistic studies of cytochrome oxidases because of the proliferation of kinetic and spectroscopic studies which use CO photodissociation to initiate redox activity with 02 (9-13). If photodissociated CO binds to another redox center of the enzyme and remains