This work was performed during tenure of Career Development Award GM-K3-31.213.1 Abbreviations used: epr, electron paramagnetic resonance;FMN, riboflavin 5'-phosphate; TPNH, reduced triphosphopyridine nucleotide; FAD, FADH, FADHi, oxidized, semiquinoid, and fully reduced flavin-adenine dinucleotide; TPN+, oxidized triphosphopyridine nucleotide.cals, which are very readily distinguished on the basis of their optical absorption properties. With one enzyme, glucose oxidase, both types of radical are found, the concentrations depending on the pH. These results suggest that the two different spectral species may be due to the neutral and anionic radical forms of the flavin coenzyme prosthetic groups.et al., 1966) and Azotobacter flavoprotein (Beinert, 1965). Again this long wavelength absorption has been found to be well correlated with an epr-detectable free radical, and experimental extinction coefficients in the range 3000-5000 1. mole-1 cm-1 have been found at 570 µ. In the case of two other flavoproteins, d-

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Upon reaction of cytochrome oxidase with hydrogen peroxide, the spectral changes are complete, with slightly less than 1 equiv of hydrogen peroxide per cytochrome oxidase. At pH 8 the product is a mixture of the P and F forms, while at pH 6 the product is exclusively the F form. These data are inconsistent with current interpretations of the structure of compounds P and F. Two stable radical species are detected by EPR; the relative amounts of these species are pH dependent. The MCD spectra of pure P and F are reported. It is suggested that compound F is a hydrogen peroxide adduct of cytochrome oxidase with cytochrome a3 in the low-spin state and that compound P is an oxyferryl state of cytochrome alpha 3 in support of the recent Raman data of Proshlyakov et al. [(1994) J. Biol. Chem. 269, 29385-29388]. We also suggest that copper B is in the trivalent state in compound P.

The coupling between the peroxidase and cyclooxygenase activities of prostaglandin H synthase (PGHS) has been proposed to be mediated by a critical tyrosyl radical through a branched chain mechanism (Dietz, R., Nastainczyk, W., and Ruf, H. H. (1988) Eur. J. Biochem. 171, 321-328). In this study, we have examined the ability of PGHS isoform-1 (PGHS-1) tyrosyl radicals to react with arachidonate. Anaerobic addition of arachidonate following formation of the peroxide-induced wide doublet or wide singlet tyrosyl radical led to disappearance of the tyrosyl radicals and emergence of a new EPR signal, which is distinct from known PGHS-1 tyrosyl radicals. The new radical was clearly derived from arachidonate because its EPR line shape changed when 5,6,8,9,11,12,14,15-octadeuterated arachidonate was used. Subsequent addition of oxygen to samples containing the fatty acyl radical resulted in regeneration of tyrosyl radical EPR. In contrast, the peroxide-generated tyrosyl radical in indomethacin-treated PGHS-1 (a narrow singlet) failed to react with arachidonate, consistent with the cyclooxygenase inhibition by indomethacin. These results indicate that the peroxide-generated wide doublet and wide singlet tyrosyl radicals serve as immediate oxidants of arachidonate bound at the cyclooxygenase active site to form a carbon-centered fatty acyl radical, which reacts with oxygen to form a hydroperoxide. These observations represent the first direct evidence of chemical coupling between the peroxidase reaction and arachidonate oxygenation in PGHS-1 and support the proposed role for a tyrosyl radical in cyclooxygenase catalysis.

The kinetics of the flash-induced photodissociation and rebinding of carbon monoxide in cytochrome aa3-CO have been studied by time-resolved infrared (TRIR) and transient ultraviolet-visible (UV-vis) spectroscopy at room temperature and by Fourier transform infrared (FTIR) spectroscopy at low temperature. The binding of photodissociated CO to CuB+ at room temperature is conclusively established by the TRIR absorption at 2061 cm-1 due to the C-O stretching mode of the CuB(+)-CO complex. These measurements yield a first-order rate constant of (4.7 +/- 0.6) x 10(5) s-1 (t1/2 = 1.5 +/- 0.2 microseconds) for the dissociation of CO from the CuB(+)-CO complex into solution. The rate of rebinding of flash-photodissociated CO to cytochrome a(3)2+ exhibits saturation kinetics at [CO] > 1 mM due to a preequilibrium between CO in solution and the CuB(+)-CO complex (K1 = 87 M-1), followed by transfer of CO to cytochrome a(3)2+ (k2 = 1030 s-1). The CO transfer from CuB to Fe alpha 3 was followed by CO-FTIR between 158 and 179 K and by UV-vis at room temperature. The activation parameters over the temperature range 140-300 K are delta H++ = 10.0 kcal mol-1 and delta S++ = -12.0 cal mol-1 K-1. The value of delta H++ is temperature independent over this range; i.e., delta Cp++ = 0 for CO transfer. Rapid events following photodissociation and preceding rebinding of CO to cytochrome a(3)2+ were observed. An increase in the alpha-band of cytochrome a3 near 615 nm (t1/2 ca. 6 ps) follows the initial femtosecond time-scale events accompanying photodissociation. Subsequently, a decrease is observed in the alpha-band absorbance (t1/2 approximately 1 microsecond) to a value typical of unliganded cytochrome a3. This latter absorbance change appears to occur simultaneously with the loss of CO by CuB+. We ascribe these observations to structural changes at the cytochrome a3 induced by the formation and dissociation of the CuB(+)-CO complex. We suggest that the picosecond binding of photodissociated CO to CuB triggers the release of a ligand L from CuB. We infer that L then binds to cytochrome a3 on the distal side and that this process is directly responsible for the observed alpha-band absorbance changes. We have previously suggested that the transfer of L produces a transient five-coordinate high-spin cytochrome a3 species where the proximal histidine has been replaced by L. When CO binds to the enzyme from solution, these processes are reversed.(ABSTRACT TRUNCATED AT 400 WORDS)