Lars-Erik Andréasson

Properties and function of the two hemes in Pseudomonas cytochrome c peroxidase

The oxidation-reduction potentials of the two c-type hemes of Pseudomonas aeruginosa cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase EC 1.11.1.5) have been determined and found to be widely different, about +320 and -330 mV, respectively. The EPR spectrum at temperatures below 77 K reveals only low-spin signals (gz 3.24 and 2.93), whereas optical spectra at room temperature indicate the presence of one high-spin and one low-spin heme in the enzyme. Optical absorption spectra of both resting and half-reduced enzyme at 77 K lack features of a high-spin compound. It is concluded that the heme ligand arrangement changes on cooling from 298 to 77 K with a concomitant change in the spin state. The active form of the peroxidase is the half-reduced enzyme, in which one heme is in the ferrous and the other in the ferric state (low-spin below 77 K with gz 2.84). Reaction of the half-reduced enzyme with hydrogen peroxide forms Compound I with the hemes predominantly in the ferric (gz 3.15) and the ferryl states. Compound I has a half-life of several seconds and is converted into Compound II apparently having a ferric-ferric structure, characterized by an EPR peak at g 3.6 with unusual temperature and relaxation behavior. Rapid-freeze experiments showed that Compound II is formed in a one-electron reduction of Compound I. The rates of formation of both compounds are consistent with the notion that they are involved in the catalytic cycle.

Ca2+ depletion modifies the electron transfer on both donor and acceptor sides in Photosystem II from spinach

Ca2+ depletion of Photosystem II from spinach results in reversible retardation of electron transfer on both donor and acceptor sides. On the donor side, a decrease of the electron transfer rate from TyrZ results in an enhanced charge recombination between the oxidized primary donor, P680+, and the reduced acceptor quinone, QA−, which in turn leads to a decrease in the amplitude of the fluorescence yield. In addition, slow electron transfer from the manganese cluster in the dark-stable S2 state results in the appearance of a transient EPR signal from TyrZox which decays with half-times of 600 ms and 5 s. On the acceptor side, the disappearance of the 400 μs decay transient in the fluorescence yield indicates that the electron transfer from QA− to QB has been severely inhibited. These results suggests that removal of a Ca2+ ion from the donor side in PS II, which results in the inhibition of oxygen evolution and in the appearance of an EPR signal in the S′3 state leads to structural changes which are transmitted to the acceptor side. The strikingly similar behavior after depletion of Ca2+ of the TyrZox EPR signal and the split radical signal from the S′3 state suggests that both signals involves the same oxidized amino acid residue, TyrZox. The absence of large effects on the EPR properties of the non-heme iron suggests that the structural changes on the acceptor side are subtle in nature. Chemical modification of histidine results in inhibition of QA− to QB electron transfer and to changes in the magnetic properties of the oxidized non-heme iron but only to minor perturbations of the donor-side. This suggests that histidine, susceptible to chemical modification, is located mainly on the acceptor side of PS II.

Studies of the slowly exchanging chloride in Photosystem II of higher plants.

Abstract36Cl- was used to study the slow exchange of chloride at a binding site associated with Photosystem II (PS II). When PS II membranes were labeled with different concentrations of 36Cl-, saturation of binding at about I chloride/PS II was observed. The rate of binding showed a clear dependence on the concentration of chloride approaching a limiting value of about 3·10-4 s-1 at high concentrations, similar to the rate of release of chloride from labeled membranes. These rates were close to that found earlier for the release of chloride from PS II membranes isolated from spinach grown on 36Cl-, which suggests that we are observing the same site for chloride binding. The similarity between the limiting rate of binding and the rate of release of chloride suggests that the exchange of chloride with the surrounding medium is controlled by an intramolecular process. The binding of chloride showed a pH-dependence with an apparent pKa of 7.5 and was very sensitive to the presence of the extrinsic polypeptides at the PS II donor side. The binding of chloride was competitively inhibited by a few other anions, notably Br- and NO3-. The slowly exchanging Cl- did not show any significant correlation with oxygen evolution rate or yield of EPR signals from the S2 state. Our studies indicate that removal of the slowly exchanging chloride lowers the stability of PS II as indicated by the loss of oxygen evolution activity and S2 state EPR signals.

The reaction of ferrocytochrome c with cytochrome oxidase: A new look

The reaction of purified cytochrome oxidase (ferrocytochrome c: Oa oxidoreductase, E.C. 1.9.3.1) with its natural substrate, cytochrome c, has for obvious reasons received a good deal of attention (see, for example, [l] for a review). Kinetic measurements in a stopped-flow apparatus of the direct reaction between the oxidized form of the oxidase and c2+ have, however, only been carried out to a limited extent, the most thorough study probably being that of Gibson et al. [2] . In “anaerobic” (about 0.1 PM 0,) experiments these authors found an initial rapid reaction, which they ascribed to the reduction of cytochrome a and one of the copper components, followed by slower changes. The second phase of the reaction was believed to be slow because of the formation of a complex between u2+ and c3+, in which electrons are not readily transferred to a?. For a number of reasons we have found it interesting to repeat and extend the type of stopped-flow measurements reported by Gibson et al. [2] . First, these authors did not include observations on the 830nm band generally associated with one of the copper components of the enzyme (see [l] ). Second, we have recently incorporated [3,4] in our stoppedflow technique certain improvements which are deemed important for a complete description of the reaction. Thus, the auxiliary equipment of our apparatus allows accurate direct measurements of absolute, and not only difference, absorbance values. In addition, we can keep the concentration of O2 in the system below 0.1 /LM

The interaction of ammonia with the photosynthetic oxygen-evolving system

Abstract The reaction of ammonia with the oxygen-evolving system was investigated using EPR. Two sites with distinct binding properties were found. One site, previously known to be responsible for the modification by ammonia of the multiline EPR signal from the S2 state and believed to be accessible in this state only, was found to bind ammonia also in the S1 state although weaker. The second binding site, identified by the effect of bound ammonia on the shape and position of the g = 4.1 EPR signal, was also found to be accessible in both the S1 and S2 states. The apparent dissociation constants for ammonia at the two sites in the S1 and S2 states were determined. In neither state did the binding the ammonia account for the observed inhibition of oxygen evolution, suggesting that binding to other S states plays an important role in the inhibition. Chloride, which is known to interfere with ammonia-induced inhibition of oxygen evolution, was found to compete with ammonia at the site associated with the modification of the g = 4.1 EPR signal. The broadening of the hyperfine lines of the multiline EPR signal, seen in the presence of 17O-labeled water, was still observed after the modification of the signal by ammonia. This indicates that ammonia has not completely displaced water bound to the catalytic site in the S2 state. The results of the binding studies are interpreted in terms of a two state — two site model, where the two states are identified by their EPR signals, the multiline and the g = 4.1 signal, respectively, and the two sites identified by the effects of ammonia on these signals and where the equilibrium between the two states is regulated by the binding of ligands to the sites.

The nature of the Fe(III) EPR signal from the acceptor-side iron in photosystem II

The EPR spectrum at both X‐ and S‐band (3.94 GHz) of the oxidized acceptor‐side iron in photosystem II from spinach shows two absorption‐type peaks at g = 8.0 and 5.6. The intensities of these peaks have been measured at X‐band in the temperature range 2–10 K. All results can be fully described assuming that the EPR spectrum arises from high‐spin Fe(III) with D = 1.0±0.3 cm−1 and E/D = 0.10 ±0.01. Quantifications show that the spectrum in our case represents 0.4–0.5 Fe(III) per reaction center. The EPR parameters are consistent with the iron having bicarbonate and/or tyrosine as ligands in addition to four imidazoles.

A comparison between the multiline EPR signals of spinach and Anacystis nidulans and their temperature dependence

The multiline EPR signals arising from manganese in the S2 state of the oxygen‐evolving system of spinach and the cyanobacterium Anacystis nidulans have very similar properties and are affected identically by NH3, suggesting that the system is highly conserved. The temperature dependence of the signal amplitude follows Curie behavior down to sub‐helium temperatures. This is in contrast to previous reports, which were taken as evidence for a tetrameric manganese cluster. Thus, it seems that it is not yet possible from EPR data alone to distinguish between this model and a dimeric structure.

Kinetics and mechanism: The steady-state rate equation for cytochrome c oxidase based on a minimal Kinetic Scheme

: A minimal catalytic cycle for cytochrome c oxidase has been suggested, and the steady-state kinetic equation for this mechanism has been derived. This equation has been used to simulate experimental data for the pH dependence of the steady-state kinetic parameters, kcat and Km. In the simulations the rate constants for binding and dissociation of cytochrome c and for two internal electron-transfer steps have been allowed to vary, whereas fixed experimental values (for pH 7.4) have been used for the other rate constants. The results show that the dissociation of the product, ferricytochrome c, cannot be rate-limiting under all conditions, but that intramolecular electron-transfer steps also limit the rate. They also demonstrate that Km can differ considerably from the dissociation constant for the cytochrome c-oxidase complex. Published values for the rate constant for the dissociation of ferricytochrome c are too small to account for the steady-state rates. It is suggested that, at high concentrations, ferryocytochrome c transfers an electron to a cytochrome c molecule which remains bound to the oxidase. This can also explain the nonhyperbolic kinetics, which is observed at low substrate concentrations.A minimal catalytic cycle for cytochrome c oxidase has been suggested, and the steady-state kinetic equation for this mechanism has been derived. This equation has been used to simulate experimental data for the pH dependence of the steady-state kinetic parameters, kcat and Km. In the simulations the rate constants for binding and dissociation of cytochrome c and for two internal electron-transfer steps have been allowed to vary, whereas fixed experimental values (for pH 7.4) have been used for the other rate constants. The results show that the dissociation of the product, ferricytochrome c, cannot be rate-limiting under all conditions, but that intramolecular electron-transfer steps also limit the rate. They also demonstrate that Km can differ considerably from the dissociation constant for the cytochrome c-oxidase complex. Published values for the rate constant for the dissociation of ferricytochrome c are too small to account for the steady-state rates. It is suggested that, at high concentrations, ferryocytochrome c transfers an electron to a cytochrome c molecule which remains bound to the oxidase. This can also explain the nonhyperbolic kinetics, which is observed at low substrate concentrations.

Isnitrogen liganded to manganese in the photosynthetic oxygen-evolving system? EPR studies after isotopic replacement with 15N

The nature of the ligands to manganese in the photosynthetic oxygen-evolving system was investigated by the effect of isotopic substitution with 15N on the spectral properties of the multiline EPR signal of the S2 state. No changes were observed in the general shape of the signal or in the linewidth. The results show that the resolved fine structure in the multiline signal cannot be explained as nitrogen superhyperfine structure but most likely arises from manganese hyperfine interaction. The large difference between the highest value for the nitrogen coupling constant consistent with the results and that earlier observed in a binuclear, antiferromagnetically coupled manganese model complex strongly favors non-nitrogen ligands such as oxygen in carboxylic amino acid side chains.

The inhibition of photosynthetic oxygen evolution by ammonia probed by EPR

Abstract EPR was used to study the binding of NH 3 to the photosynthetic O 2 -evolving center, NH 3 -treated, Ca 2+ -depleted Photosystem II (PS II) membranes exposed to continuous light at 250 K showed a 10 mT-wide asymmetric EPR signal, centered around g = 2. When dark-adapted material was illuminated with a sequence of laser flashes the same signal appeared after the second flash, indicating that the g = 2 signal arises from a modified S 3 state. The signal is different from the 15–16.5 mT-wide EPR signal at g = 2 ascribed to the S 3 ′ state. Illumination of native NH 3 -treated PS II membranes with continuous light results in the appearance of an EPR signal at g = 2 with a width similar to that in Ca 2+ -depleted. NH 3 -treated membranes. The conditions for the formation of the signal and its properties suggest that it also arises from a perturbed S 3 state with NH 3 in close association with the manganese.