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A novel electrochemical system has been designed and assembled to study the kinetic activity of cytochrome c oxidase. Gold electrodes coated with 3-mercapto-1-propanol formed the surface for the physisorption of monolayers of cytochrome c and cytochrome c oxidase or a preformed cytochrome c−cytochrome c oxidase complex. The films were investigated by cyclic voltammetry at scanning at rates slow enough to permit near redox equilibrium between electrode and redox protein and hence obtain redox midpoint potentials. Cytochrome c monolayers alone displayed a reversible midpoint potential at pH 8 (Em8 vs NHE) at +240 mV, close to the native cytochrome c value observed in solution. In contrast, oxidase monolayers alone failed to support any detectable redox contact between electrode and protein, implying that the distances between the oxidase redox cofactors in the adsorbed oxidase are too far away from the electrode to promote significant electron transfer rates. However, adsorption of a preformed cytochrome c−cytochrome c oxidase complex promoted effective redox contact, demonstrating electron transfer with an apparent onset halfpoint potential at +225 mV. This effect is consistent with the mandatory requirement for cytochrome c to mediate electrons from the electrode to cytochrome c oxidase and presumably in a way reflecting the physiological pathway. Cyclic voltammetric measurements arranged to determine the rates of electron transfer between electrode and the complex showed that at scan rates up to 50 mV/s, extraordinary kinetic turnover is displayed attributable to the catalysis of oxygen reduction. Thus it is established that the protein complex can be assembled and enable the natural mediation of electron transfer from the electrode by cytochrome c to the enzyme at a rate fast enough for catalysis to be observed and controlled.

We have investigated the cleavage of the bond between the heme iron and the proximal heme ligand, His388, in ferrous ovine prostaglandin endoperoxide H synthase-1 (oPGHS-1) on nitric oxide (NO) binding by using resonance Raman spectroscopy. The Fe−NO and N−O vibrations are observed at 526 and 1667 cm^-1, respectively, which is indicative of a five-coordinated, heme−NO complex. We also observed NO photolysis under low-power, continuous wave conditions, which reflects nongeminate rebinding of NO to the heme cofactor on the millisecond time scale. Furthermore, we attempted to make a six-coordinated heme−NO complex by adding imidazole (Im) to ferrous oPGHS-1-NO. However, ferrous oPGHS-1-NO remained five-coordinated even in the presence of 500 mM Im. The heme−NO complex remains bound in the heme pocket after Fe−His bond cleavage, and subsequent protein backbone movement is relatively small. Finally, we compared the structures of several heme proteins that form either five- or six-coordinated complexes with NO. Inspection of the heme-proximal His geometry did not reveal any common structural parameter that may be critical for Fe−His bond cleavage. However, the Fe−His stretching frequency [ν(Fe−His)] shows a good correlation with Fe−His bond cleavage and is modulated by hydrogen bonding to the Nδ proton of the His. We propose that the strength of this hydrogen bond is the primary factor determining Fe−His bond cleavage:  a strong hydrogen bond gives the His imidazolate character, which allows it to compensate for the NO trans ligand effect and to avoid Fe−His bond cleavage.

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).

Using density functional theory (DFT) techniques, we have investigated possible conformers of the radical anions of plastoquinone (psQ), ubiquinone (ubQ), and menaquinone (mnQ), which are formed in the reaction centers of photosynthetic bacteria, blue-green bacteria, and green plants. Replacing the hydrocarbon tail connected to the quinone ring by an ethyl group, we have computed the rotational potential energy surfaces for psQ^- and ubQ^-. Our results show that in the absence of environmental effects both systems have global minima for near-perpendicular orientations of the γ-carbon relative to the quinone ring. For psQ^-, however, a low-lying local minimum is also observed for an in-plane arrangement with Cγ pointing away from the O4 oxygen. These differences in head-to-tail rotational energy surfaces may explain the experimentally observed differences in β-proton hyperfine couplings of psQ^- vs ubQ^- and mnQ^-, and their corresponding model compounds. By replacing the C6 methyl group in ubQ and mnQ by hydrogen, or the C6 hydrogen in psQ by methyl, we show that the crucial factor determining the rotational arrangements of the quinones in biological systems (planar psQ in green plants; perpendicular ubQ and mnQ in bacteria) is the presence or absence of this methyl group. The computed barrier height to rotation in ubQ^-, ca. 6 kcal/mol, and the β-proton hyperfine coupling constants for the planar vs perpendicular arrengements are in excellent accord with experimental data. Finally, we show that the methoxy group at the C2 position in ubiquinone displays a conformational preference as a result of the electron addition process, which may effect the hydrogen bonding pattern and hence promote the electron-transfer processes.

The early suggestion by Lozier and Butler (Photochem.Photobiol.17, 133–137 (1973)) that EPR Signal II arises from radicals associated with the water-splitting process in PSII has been confirmed and extended over the intervening years. Recent work has identified the Signal II radicals, and , with plastosemiquinone cation species. In the experiments presented here we have used ENDOR spectroscopy and D2O/H2O exchange to characterize these paramagnets in more detail. The ENDOR matrix region, which arises from protons which interact weakly with the unpaired electron spin, is well-resolved at 4 K and at least seven resonances are apparent. A number of hyperfine couplings in the 3–8 MHz range are observed and are suggested to arise from methyl or hydroxyl protons which occur as substituents on the plastosemiquinone cation ring or from amino acid protons hydrogen-bonded to the 1,4-hydroxyl groups. Orientation selection experiments are consistent with these possibilities. D2O/H2O exchange shows that the D+/Z+ site is accessible to solvent. However, the exchange occurs slowly and is not complete even after 72 hours which suggests that the free radicals are functionally isolated from solvent water.

Photosynthetic oxygen evolution takes place in the thylakoid protein complex known as photosystem II. The reaction center core of this photosystem, where photochemistry occurs, is a heterodimer of homologous polypeptides called D1 and D2. Besides chlorophyll and quinone, photosystem II contains other organic cofactors, including two known as Z and D. Z transfers electrons from the site of water oxidation to the oxidized reaction center primary donor, P+.680, while D+. gives rise to the dark-stable EPR spectrum known as signal II. D+. has recently been shown to be a tyrosine radical. Z is probably a second tyrosine located in a similar environment. Indirect evidence indicates that Z and D are associated with the D1 and D2 polypeptides, respectively. To identify the specific tyrosine residue corresponding to D, we have changed Tyr-160 of the D2 polypeptide to phenylalanine by site-directed mutagenesis of a psbD gene in the cyanobacterium Synechocystis 6803. The resulting mutant grows photosynthetically, but it lacks the EPR signal of D+.. We conclude that D is Tyr-160 of the D2 polypeptide. We suggest that the C2 symmetry in photosystem II extends beyond P680 to its immediate electron donor and conclude that Z is Tyr-161 of the D1 polypeptide.

In addition to the reaction-center chlorophyll, at least two other organic cofactors are involved in the photosynthetic oxygen-evolution process. One of these cofactors, called "Z," transfers electrons from the site of water oxidation to the reaction center of photosystem II. The other species, "D," has an uncertain function but gives rise to the stable EPR signal known as signal II. Z+. and D+. have identical EPR spectra and are generally assumed to arise from species with the same chemical structure. Results from a variety of experiments have suggested that Z and D are plastoquinones or plastoquinone derivatives. In general, however, the evidence to support this assignment is indirect. To address this situation, we have developed more direct methods to assign the structure of the Z+./D+. radicals. By selective in vivo deuteration of the methyl groups of plastoquinone in cyanobacteria, we show that hyperfine couplings from the methyl protons cannot be responsible for the partially resolved structure seen in the D+. EPR spectrum. That is, we verify by extraction and mass spectrometry that quinones are labeled in algae fed deuterated methionine, but no change is observed in the line shape of signal II. Considering the spectral properties of the D+. radical, a tyrosine origin is a reasonable alternative. In a second series of experiments, we have found that deuteration of tyrosine does indeed narrow the D+. signal. Extraction and mass spectral analysis of the quinones in these cultures show that they are not labeled by tyrosine. These results eliminate a plastoquinone origin for D+.; we conclude instead that D+., and most likely Z+., are tyrosine radicals.

A model is proposed for the active center of cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) in which cytochrome a is a low-spin ferrihemoprotein and cytochrome a3 is a high-spin ferrihemoprotein antiferromagnetically coupled to one of the two Cu2+ ions present in the enzyme. It is further proposed that reduction is accompanied by a conformational change in the enzyme thus exposing the sixth coordination site of cytochrome a3 to ligands. With this model it is possible to account for a variety of outstanding observations including the results of magnetic circular dichroism, Mossbauer, and electron paramagnetic resonance spectroscopies, as well as magnetic susceptibility measurements.