Electron paramagnetic resonance and electron nuclear double resonance spectroscopic identification and characterization of the tyrosyl radicals in prostaglandin H synthase 1.

The tyrosyl radicals generated in reactions of ethyl hydrogen peroxide with both native and indomethacin-pretreated prostaglandin H synthase 1 (PGHS-1) were examined by low-temperature electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies. In the reaction of peroxide with the native enzyme at 0 degrees C, the tyrosyl radical EPR signal underwent a continuous reduction in line width and lost intensity as the incubation time increased, changing from an initial, 35-G wide doublet to a wide singlet of slightly smaller line width and finally to a 25-G narrow singlet. The 25-G narrow singlet produced by self-inactivation was distinctly broader than the 22-G narrow singlet obtained by indomethacin treatment. Analysis of the narrow singlet EPR spectra of self-inactivated and indomethacin-pretreated enzymes suggests that they reflect conformationally distinct tyrosyl radicals. ENDOR spectroscopy allowed more detailed characterization by providing hyperfine couplings for ring and methylene protons. These results establish that the wide doublet and the 22-G narrow singlet EPR signals arise from tyrosyl radicals with different side-chain conformations. The wide-singlet ENDOR spectrum, however, is best accounted for as a mixture of native wide-doublet and self-inactivated 25-G narrow-singlet species, consistent with an earlier EPR study [DeGray et al. (1992) J. Biol. Chem. 267, 23583-23588]. We conclude that a tyrosyl residue other than the catalytically essential Y385 species is most likely responsible for the indomethacin-inhibited, narrow-singlet spectrum. Thus, this inhibitor may function by redirecting radical formation to a catalytically inactive side chain. Either radical migration or conformational relaxation at Y385 produces the 25-G narrow singlet during self-inactivation. Our ENDOR data also indicate that the catalytically active, wide-doublet species is not hydrogen bonded, which may enhance its reactivity toward the fatty-acid substrate bound nearby.

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. I. Biochemical, spectral, and kinetic characterization of surface mutants in subunit ii of Rhodobacter sphaeroides cytochrome aa(3).

To determine the interaction site for cytochrome c (Cc) on cytochrome c oxidase (CcO), a number of conserved carboxyl residues in subunit II of Rhodobacter sphaeroides CcO were mutated to neutral forms. A highly conserved tryptophan, Trp(143), was also mutated to phenylalanine and alanine. Spectroscopic and metal analyses of the surface carboxyl mutants revealed no overall structural changes. The double mutants D188Q/E189N and D151Q/E152N exhibit similar steady-state kinetic behavior as wild-type oxidase with horse Cc and R. sphaeroides Cc(2), showing that these residues are not involved in Cc binding. The single mutants E148Q, E157Q, D195N, and D214N have decreased activities and increased K(m) values, indicating they contribute to the Cc:CcO interface. However, their reactions with horse and R. sphaeroides Cc are different, as expected from the different distribution of surface lysines on these cytochromes c. Mutations at Trp(143) severely inhibit activity without changing the K(m) for Cc or disturbing the adjacent Cu(A) center. From these data, we identify a Cc binding area on CcO with Trp(143) and Asp(214) close to the site of electron transfer and Glu(148), Glu(157), and Asp(195) providing electrostatic guidance. The results are completely consistent with time-resolved kinetic measurements (Wang, K., Zhen, Y., Sadoski, R., Grinnell, S., Geren, L., Ferguson-Miller, S., Durham, B., and Millett, F. (1999) J. Biol. Chem. 274, 38042-38050) and computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).

From water to oxygen and back again: mechanistic similarities in the enzymatic redox conversions between water and dioxygen.

We propose that the interconversions of water and oxygen are catalyzed by the transition metal ions of Photosystem II and cytochrome c oxidase in remarkably similar ways. Oxygen-oxygen bond formation and cleavage occurs between two oxygen atoms that are bound as terminal ligands to two redox-active metal ions. Hydrogen atom transfer to or from a tyrosine residue is an essential component of the processes in both enzymes.

Manganese and tyrosyl radical function in photosynthetic oxygen evolution.

Photosystem II catalyzes the photosynthetic oxidation of water to O2. The structural and functional basis for this remarkable process is emerging. The catalytic site contains a tetramanganese cluster, calcium, chloride and a redox-active tyrosine organized so as to promote electroneutral hydrogen atom abstraction from manganese-bound substrate water by the tyrosyl radical. Recent work is assessed within the framework of this model for the water oxidizing process.

A metalloradical mechanism for the generation of oxygen from water in photosynthesis.

In plants and algae, photosystem II uses light energy to oxidize water to oxygen at a metalloradical site that comprises a tetranuclear manganese cluster and a tyrosyl radical. A model is proposed whereby the tyrosyl radical functions by abstracting hydrogen atoms from substrate water bound as terminal ligands to two of the four manganese ions. Molecular oxygen is produced in the final step in which hydrogen atom transfer and oxygen-oxygen bond formation occur together in a concerted reaction. This mechanism establishes clear analogies between photosynthetic water oxidation and amino acid radical function in other enzymatic reactions.

An examination of the source of the tyrosyl radical in ovine prostaglandin endoperoxide synthase-1.

A tyrosyl radical, which may initiate the cyclooxygenase reaction, has been detected in prostaglandin H synthase by electron paramagnetic resonance spectroscopy. In the crystal structure of ovine prostaglandin H synthase-1, Tyr348 and Tyr385 are in close proximity to the heme. We mutated these residues to phenylalanine to test for their involvement in tyrosyl radical formation. Native enzyme formed a tyrosyl radical centered at g = 2.0036 with a width of 28 gauss. The Y348F mutant formed a singlet signal similar to that of native enzyme with a width of 28 gauss (g = 2.0039). In contrast, the radical signals seen with the Y385F and Y348F/Y385F mutants were 23 gauss (g = 2.004) and 22 gauss (g = 2.0037). In short, tyrosyl radicals are formed even in the absence of both Tyr348 and Tyr385. In Y345F containing mutants, a cluster of aromatic amino acids which surrounds the heme group may provide an alternate pathway for electron abstraction from a more distant tyrosine, yielding a narrow tyrosyl radical signal.

A hydrogen-atom abstraction model for the function of YZ in photosynthetic oxygen evolution.

Recent magnetic-resonance work on YZ suggests that this species exhibits considerable motional flexibility in its functional site and that its phenol oxygen is not involved in a well-ordered hydrogen-bond interaction (Tang et al., submitted; Tommos et al., in press). Both of these observations are inconsistent with a simple electron-transfer function for this radical in photosynthetic water oxidation. By considering the roles of catalytically active amino acid radicals in other enzymes and recent data on the water-oxidation process in Photosystem II, we rationalize these observations by suggesting that YZ functions to abstract hydrogen atoms from aquo- and hydroxy-bound managanese ions in the (Mn)4 cluster on each S-state transition. The hydrogen-atom abstraction process may occur either by sequential or concerted kinetic pathways. Within this model, the (Mn)4/YZ center forms a single catalytic center that comprises the Oxygen Evolving Complex in Photosystem II.

Characterization of a tyrosyl radical in prostaglandin endoperoxide synthase-2.

Two different isoforms of prostaglandin H synthase, prostaglandin H synthase-1 and prostaglandin H synthase-2, have been identified. Both isozymes catalyze both cyclooxygenase and peroxidase reactions. Residues identified as being essential for catalysis by ovine prostaglandin endoperoxide H synthase-1 are all conserved in prostaglandin H synthase-2. This suggests that the enzymic reaction mechanisms are fundamentally the same for both isozymes. A tyrosyl radical, which may initiate the cyclooxygenase reaction, is detected by electron paramagnetic resonance spectroscopy after addition of arachidonic acid or hydroperoxides to ovine prostaglandin H synthase-1. We report here that human prostaglandin H synthase-2 also generates a tyrosyl radical centered at g = 2.0040 with a width of 29 gauss, similar to prostaglandin H synthase-1. This is the first spectral evidence that the two isoforms are similar mechanistically.

Protein-tyrosyl radical interactions in photosystem II studied by electron spin resonance and electron nuclear double resonance spectroscopy: comparison with ribonucleotide reductase and in vitro tyrosine.

The stable tyrosine radical in photosystem II, YD*, has been studied by ESR and ENDOR spectroscopies to obtain proton hyperfine coupling constants from which the electron spin density distribution can be deduced. Simulations of six previously published ESR spectra of PSII (one at Q band; five at X band, of which two were after specific deuteration and two others were of oriented membranes) can be achieved by using a single set of magnetic parameters that includes anisotropic proton hyperfine tensors, an anisotropic g tensor, and noncoincident axis systems for the g and A tensors. From the spectral simulation of the oriented samples, the orientation of the phenol head group of YD* with respect to the membrane plane has been determined. A similar orientation for YZ*, the redox-active tyrosine in PSII that mediates electron transfer between P680 and the oxygen-evolving complex, is expected. ENDOR spectra of YD* in PSII preparations from spinach and Synechocystis support the set of hyperfine coupling constants but indicate that small differences between the two species exist. Comparison with the results of spectral simulations for tyrosyl radicals in ribonucleotide reductase from prokaryotes or eukaryotes and with in vitro radicals indicates that the spin density distribution remains that of an odd-alternant radical but that interactions with the protein can shift spin density within this basic pattern. The largest changes in spin density occur at the tyrosine phenol oxygen and at the ring carbon para to the oxygen, which indicates that mechanisms exist in the protein environment for fine-tuning the chemical and redox properties of the radical species.

Mn(2+) reduces Yz (+) in manganese-depleted Photosystem II preparations.

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn(2+). Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Yz (+), the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Yz (+) reduction by benzidine was a linear function of benzidine concentration. The rate of Yz (+) reduction by Mn(2+) at pH 6 increased linearly at low Mn(2+) concentrations and reached a maximum at the Mn(2+) concentrations equal to several times the reaction center concentration. The rate was inhibited by K(+), Ca(2+) and Mg(2+). These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Yz (+) reduction at pH 7.5 was biphasic with a fast 400 µs phase that suggests binding of Mn(2+) near Yz (+) at a site that may be one of the native manganese binding sites.