Graham Palmer

The reduced minus oxidized difference spectra of cytochromes a and a3.

We have re-investigated the reduced minus oxidized difference spectra of the two heme centers of cytochrome oxidase, cytochromes a and a3. In contrast to data obtained in an earlier study (Vanneste, W.H. (1966) Biochemistry 5, 838-848), we find that the spectrum for cytochrome a3 agrees with that found with a 5-coordinate high-spin heme A model compound. Small but significant additional differences are noted for both heme centers.

Evidence for N coordination to Fe in the [2Fe-2S] center in yeast mitochondrial complex III Comparison with similar findings for analogous bacterial [2Fe-2S] proteins

Yeast mitochondrial complex III contains a subunit with a [2Fe‐2S] cluster (the Rieske center) that has unusual physical and chemical properties. For apparently similar centers isolated from bacteria, it has been shown by electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) measurements that these [2Fe‐2S] centers are coordinated by at least one and probably two nitrogen ligands. This work describes similar ENDOR and ESEEM studies on the intact mitochondrial complex. We find that this [2Fe‐2S] cluster exhibits ESEEM and ENDOR properties that appear to be indistinguishable from those observed with the isolated bacterial systems. Furthermore, changes in EPR lineshape that occur as complex III is progressively reduced are not accompanied by any changes in the nitrogen coupling parameters. This spectroscopic evidence for nitrogen coordination is supported by published sequence data on four Rieske iron‐sulfur subunits. It seems likely that this is a general characteristic of such [2Fe‐2S] redox active centers.

Rapid Kinetics of Tyrosyl Radical Formation and Heme Redox State Changes in Prostaglandin H Synthase-1 and -2

Hydroperoxide-induced tyrosyl radicals are putative intermediates in cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2. Rapid-freeze EPR and stopped-flow were used to characterize tyrosyl radical kinetics in PGHS-1 and -2 reacted with ethyl hydrogen peroxide. In PGHS-1, a wide doublet tyrosyl radical (34–35 G) was formed by 4 ms, followed by transition to a wide singlet (33–34 G); changes in total radical intensity paralleled those of Intermediate II absorbance during both formation and decay phases. In PGHS-2, some wide doublet (30 G) was present at early time points, but transition to wide singlet (29 G) was complete by 50 ms. In contrast to PGHS-1, only the formation kinetics of the PGHS-2 tyrosyl radical matched the Intermediate II absorbance kinetics. Indomethacin-treated PGHS-1 and nimesulide-treated PGHS-2 rapidly formed narrow singlet EPR (25–26 G in PGHS-1; 21 G in PGHS-2), and the same line shapes persisted throughout the reactions. Radical intensity paralleled Intermediate II absorbance throughout the indomethacin-treated PGHS-1 reaction. For nimesulide-treated PGHS-2, radical formed in concert with Intermediate II, but later persisted while Intermediate II relaxed. These results substantiate the kinetic competence of a tyrosyl radical as the catalytic intermediate for both PGHS isoforms and also indicate that the heme redox state becomes uncoupled from the tyrosyl radical in PGHS-2.

CHARACTERIZATION OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE AND ITS REACTION WITH LIGAND BY ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY

Electron paramagnetic resonance was used to characterize the heme structure of resting endothelial nitric-oxide synthase (eNOS), eNOS devoid of its myristoylation site (G2A mutant), and their heme complexes formed with 16 different ligands. Resting eNOS and the G2A mutant have a mixture of low spin and high spin P450-heme with widely different relaxation behavior and a stable flavin semiquinone radical identified by EPR as a neutral radical. This flavin radical showed efficient electron spin relaxation as a consequence of dipolar interaction with the heme center; P1/2 is independent of Ca2+-calmodulin and tetrahydrobiopterin. Seven of the 16 ligands led to the formation of low spin heme complexes. In order of increasing rhombicity they are pyrimidine, pyridine, thiazole, L-lysine, cyanide, imidazole, and 4-methylimidazole. These seven low spin eNOS complexes fell in a region between the P and O zones on the “truth diagram” originally derived by Blumberg and Peisach (Blumberg, W. E., and Peisach, J. (1971) in Probes and Structure and Function of Macromolecules and Membranes (Chance, B., Yonetani, T., and Mildvan, A. S., eds) Vol. 2, pp. 215-229, Academic Press, New York) and had significant overlap with complexes of chloroperoxidase. A re-definition of the P and O zones is proposed. As eNOS and chloroperoxidase lie closer than do eNOS and P450cam on the truth diagram, it implies that the distal heme environment in eNOS resembles chloroperoxidase more than P450cam. In contrast, 4-ethylpyridine, 4-methylpyrimidine, acetylguanidine, ethylguanidine, 2-aminothiazole, 2amino-4,5-dimethylthiazole, L-histidine, and 7-nitroindazole resulted in high spin heme complexes of eNOS, similar to that observed with L-arginine. This contrasting EPR behavior caused by families of ligands such as imidazole/L-histidine or thiazole/2-aminothiazole confirms the conclusion derived from parallel optical and kinetic studies. The ligands resulting in the low spin complexes bind directly to the heme iron, while their cognate ligands induce the formation of high spin complexes by indirectly perturbing the heme structure and excluding the original axial heme ligand in the resting eNOS (V. Berka, P.-F. Chen, and A.-L. Tsai (1997) J. Biol. Chem. 272, in press). The difference in EPR spectra of these high spin eNOS complexes, although subtle, are different for different homologs.

Prostaglandin H synthase: perturbation of the tyrosyl radical as a probe of anticyclooxygenase agents.

EPR spectroscopy was used to study the effects of various nonsteroidal anti-inflammatory agents on the peroxidase-related tyrosyl radical present in prostaglandin H synthase (prostaglandin endoperoxide synthase; EC 1.14.99.1). Two types of perturbation of the tyrosyl radical by these anticyclooxygenase agents were observed. In the first case, aspirin, indomethacin, ibuprofen, (S)-flurbiprofen, and (S)-naproxen converted the doublet tyrosyl EPR signal seen on reaction of the uninhibited enzyme with ethyl hydroperoxide to a singlet bearing additional partially resolved hyperfine splittings. These compounds also decreased the maximum amount of radical generated, but they did not change the kinetics of formation and decay of the tyrosyl radical. In the second case, acetaminophen and three fenamate analogs (meclofenamate, flufenamate, and mefenamate) did not perturb the EPR line shape observed after reaction with hydroperoxide but did cause a more rapid decay of the tyrosine radical species. It would appear that, despite considerable variation in structure, the nonsteroidal anti-inflammatory agents may inhibit the cyclooxygenase activity of the synthase by two basic mechanisms.

Ferric alpha-hydroxyheme bound to heme oxygenase can be converted to verdoheme by dioxygen in the absence of added reducing equivalents.

Whether or not reducing equivalents are indispensable for the conversion of ferric α-hydroxyheme bound to heme oxygenase-1 to verdoheme remains controversial (Matera, K. M., Takahashi, S., Fujii, H., Zhou, H., Ishikawa, K., Yoshimura, T., Rousseau, D. L., Yoshida, T., and Ikeda-Saito, M. (1996)J. Biol. Chem. 271, 6618–6624; Liu, Y., Moënne-Loccoz, P., Loehr, T. M., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 6906–6917). To resolve this controversy, we have prepared a ferric α-hydroxyheme-heme oxygenase-1 complex and titrated the complex with O2 under strictly anaerobic conditions. The formation of verdoheme was monitored by optical and electron spin resonance spectroscopies. Electron spin resonance spectra of the complex showed that α-hydroxyheme exists as a mixture of resonance structures composed of the iron(III) porphyrin and the iron(II) porphyrin π neutral radical. Upon addition of CO the latter species becomes dominant. The results obtained from these titration experiments indicate that α-hydroxyheme can be converted to verdoheme by an approximately equimolar amount of O2 without any requirement for exogenous electrons. The verdoheme formed from α-hydroxyheme was shown to be in the ferrous oxidation state by the addition of CO or potassium ferricyanide to the resultant verdoheme-heme oxygenase-1 complex.

Current issues in the chemistry of cytochrome c oxidase

Some contemporary issues relevant to the chemistry of mammalian cytochromec oxidase are discussed. These include the optical properties of heme A and the spectroscopic consequences of the differences in side-chain substitution compared to heme B; a common fallacy concerning the electrostatic exchange interaction between cytochromea3 and CuB; the question of the number and location of the copper components of the enzyme; and the mode of binding of ligands such as cyanide and azide.

Microcomputer tools for steady–state enzyme kinetics

Three programs useful for the investigation of steady-state kinetics have been developed. Two provide the solution to the steady-state rate equation; the first of these is a straightforward implementation of the rules developed by Chou. The second is a very efficient procedure for evaluating King-Altman diagrams and can be used for quite large mechanisms. The third program provides the numeric solution for a specific mechanism and set of initial conditions; it is well suited to extremely large models.

Spectral and Kinetic Equivalence of Oxidized Cytochrome c Oxidase as Isolated and “Activated” by Reoxidation

The spectral and kinetic characteristics of two oxidized states of bovine heart cytochrome c oxidase (CcO) have been compared. The first is the oxidized state of enzyme isolated in the fast form (O) and the second is the form that is obtained immediately after oxidation of fully reduced CcO with O2 (OH). No observable differences were found between O and OH states in: (i) the rate of anaerobic reduction of heme a3 for both the detergent-solubilized enzyme and for enzyme embedded in its natural membraneous environment, (ii) the one-electron distribution between heme a3 and CuB in the course of the full anaerobic reduction, (iii) the optical and (iv) EPR spectra. Within experimental error of these characteristics both forms are identical. Based on these observations it is concluded that the reduction potentials and the ligation states of heme a3 and CuB are the same for CcO in the O and OH states.

Reductive titration of CoQ-depleted Complex III from Baker's yeast: Evidence for an exchange-coupled complex between QH· and low-spin ferricytochrome b

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