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The Ca{sup 2+}-binding properties of photosystem II were investigated with radioactive {sup 45}Ca{sup 2+}. PS II membranes, isolated from spinach grown on a medium containing {sup 45}Ca{sup 2+}, contained 1.5 Ca{sup 2+} per PS II unit. Approximately half of the incorporated radioactivity was lost after incubation for 30 h in nonradioactive buffer. About 1 Ca{sup 2+}/PS II bound slowly to Ca{sup 2+}-depleted membranes in the presence of the extrinsic 16- and 23-kDa polypeptides in parallel with restoration of oxygen-evolving activity. The binding was heterogeneous with dissociation constants of 60 {mu}M (0.7 Ca{sup 2+}/PS II) and 1.7 mM (0.3 Ca{sup 2+}/ PS II), respectively, which could reflect different affinities of the dark-stable S-states for Ca{sup 2+}. The reactivation of oxygen-evolving activity closely followed the binding of Ca{sup 2+}, showing that a single exchangeable Ca{sup 2+} per PS II is sufficient for the water-splitting reaction to function. In PS II, depleted of the 16- and 23-kDa polypeptides, about 0.7 exchangeable Ca{sup 2+}/PS II binds with a dissociation constant of 26 {mu}M, while 0.3 Ca{sup 2+} binds with a much weaker affinity (K{sub d} > 0.5 mM). The rate of binding of Ca{sup 2+} in the absence of the two extrinsic polypeptides wasmore » significantly higher than with the polypeptides bound. The rate of dissociation of bound Ca{sup 2+} in the dark, which had a half-time of about 80 h in intact PS H, increased in the absence of the 16-and 23-kDa polypeptides and showed a further increase after the additional removal of the 33-kDa protein and manganese. The rate of dissociation was also significantly faster in weak light than in the dark. Removal of the 33-kDa donor-side polypeptide together with the two lighter ones led to a reduction in the amount of bound Ca{sup 2+}, while practically no Ca{sup 2+} bound after treatments to dissociate also the manganese of the water-oxidizing site. 34 refs., 9 figs., 2 tabs.« less

The quenching of the triplet state of Zn-cytochrome c in electrostatic complexes with cytochrome oxidase and its soluble CuA domain has been studied by laser flash photolysis. The triplet state of free Zn-cytochrome c decayed with a rate of about 200 s-1. With the oxidase, biphasic decay with rate constants of 2 x 10(5) and 2 x 10(3) s-1, respectively, was observed. At high ionic strength (I = 0.2) the decay was the same as with free Zn-cytochrome c. The quenching was also eliminated by reduction of the oxidase. The decay rate in the complex with the CuA domain was 4 x 10(4) s-1. The results are interpreted in terms of rapid electron transfer to CuA and a slower one to cytochrome a. No electron transfer products were detected, because the backward reaction is faster than the forward one. This can be explained by the high driving force (1.1 eV) for the forward electron transfer, taking the system into the inverted Marcus region. The distance in the electrostatic complex between cytochrome c and the electron acceptor, presumed to be CuA, is calculated to be 16 A.

Absorbance changes following CO dissociation by flash photolysis from mixed-valence aa3 cytochrome oxidase from Rhodobacter sphaeroides have been followed in the Soret and alpha regions. They reflect internal electron transfer in the partially reduced enzyme, and the kinetics of the reactions has been determined. As with the bovine enzyme, three kinetic phases are found with relaxation time constants at neutral pH of about 3 microseconds, 35 microseconds, and 1 ms. The first reaction phase represents electron transfer from cytochrome a3 to cytochrome a, and the extent of this reaction is about 3 times larger compared to the bovine enzyme. The energetics of the reaction has been analyzed on the basis of measurements of its temperature dependence. The reorganization energy is close to 120 kJ mol-1, and it is suggested that this rather high value is the result of changes in solvation at the cytochrome a3-CuB site. The subsequent electron transfer between cytochrome a and CuA, with a time constant of 35 microseconds, is almost activationless and has a very low reorganization energy. The final phase, with a time constant close to 1 ms at neutral pH, represents a further shift in the equilibrium between cytochrome a3 and cytochrome a, and it is limited by proton-transfer reactions. The pKa values of the groups involved are significantly shifted in the bacterial oxidase compared to the bovine one. The total extent of electron transfer in the three backflow reactions has also been determined by a comparison of the CO-recombination rates in the mixed-valence and fully reduced enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Heme-copper oxidases (HCuOs) are integral membrane proteins that catalyze the reduction of oxygen. They terminate the respiratory chain in mitochondria and most bacteria. HCuOs convert the free energy of oxygen reduction into a proton-motive force across the membrane, by pumping protons over the membrane and by using protons from the negative side of the membrane while electrons are donated from the positive side. The HCuO superfamily has been divided into the oxygen-reducing A-, B- and C-type oxidases as well as the bacterial NO reductases (NOR). Even though NORs are capable of reducing oxygen to water, their physiological roles are as NO reducers. NO reduction is performed by denitrifying bacteria, but also by pathogens that use it to break down the toxic NO produced by the immune system of the host.In the NORs, recent structural work has shown that there are possible differences in the proton transfer (PT) pathways of the NOR that uses cyt. c as electron donor (cNOR) and the NOR that uses quinol (qNOR). For qNOR a putative PT pathway from the cytoplasm was suggested, while cNOR is known to be non-electrogenic and to use periplasmic protons for NO-reduction (2NO + 2e− + 2H+ → N2O + H2O). We study the PT pathways using site-directed mutagenesis in combination with time-resolved spectroscopy during single turnovers. We show that PT in cNOR is mediated by a specific pathway from the periplasmic site. At the same time we try to engineer a proton transfer pathway from the cytoplasm in cNOR to see if we can generate an electrogenic NOR and to get more insight into the requirements for a functional PT pathway.

Wild-type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides were characterized by EPR spectroscopy. A pH-induced g 12 signal, seen previously in mammalian cytochrome oxidase and assigned to the presence of a bridging car☐yl ligand in the bimetallic cytochrome a 3 -Cu B site, is found also in the bacterial enzyme. Mutation of glutamate-286 to glutamine inactivates the enzyme but does not affect this signal, demonstrating that the car☐yl group of this residue is not the bridging ligand. Three mutants, M106Q, located one helix turn below a histidine ligand to cytochrome a , and T352A as well as F391Q, located close to the bimetallic center, are shown to affect dramatically the low-spin heme signal of cytochrome a . These mutants are essentially inactive, suggesting that these three mutations result in alterations to cytochrome a that render the oxidase non-functional.

Heme–copper oxidases (HCuOs) are the terminal components of the respiratory chain in the mitochondrial membrane or the cell membrane in many bacteria. These enzymes reduce oxygen to water and use the free energy from this reaction to maintain a proton-motive force across the membrane in which they are embedded. The heme–copper oxidases of the cbb3-type are only found in bacteria, often pathogenic ones since they have a low Km for O2, enabling the bacteria to colonize semi-anoxic environments. Cbb3-type (C) oxidases are highly divergent from the mitochondrial-like aa3-type (A) oxidases, and within the heme–copper oxidase family, cbb3 is the closest relative to the most divergent member, the bacterial nitric oxide reductase (NOR). Nitric oxide reductases reduce NO to N2O without coupling the reaction to the generation of any electrochemical proton gradient. The significant structural differences between A- and C-type heme–copper oxidases are manifested in the lack in cbb3 of most of the amino acids found to be important for proton pumping in the A-type, as well as in the different binding characteristics of ligands such as CO, O2 and NO. Investigations of the reasons for these differences at a molecular level have provided insights into the mechanism of O2 and NO reduction as well as the proton-pumping mechanism in all heme–copper oxidases. In this paper, we discuss results from these studies with the focus on the relationship between proton transfer and ligand binding and reduction. In addition, we present new data, which show that CO binding to one of the c-type hemes of CcoP is modulated by protein–lipid interactions in the membrane. These results show that the heme c-CO binding can be used as a probe of protein–membrane interactions in cbb3 oxidases, and possible physiological consequences for this behavior are discussed.

Acidianus ambivalens is a hyperthermoacidophilic archaeon which grows optimally at approximately 80 degrees C and pH 2.5. The terminal oxidase of its respiratory system is a membrane-bound quinol oxidase (cytochrome aa(3)) which belongs to the heme-copper oxidase superfamily. One difference between this quinol oxidase and a majority of the other members of this family is that it lacks the highly-conserved glutamate (Glu(I-286), E. coli ubiquinol oxidase numbering) which has been shown to play a central role in controlling the proton transfer during reaction of reduced oxidases with oxygen. In this study we have investigated the dynamics of the reaction of the reduced A. ambivalens quinol oxidase with O(2). With the purified enzyme, two kinetic phases were observed with rate constants of 1.8z.ccirf;10(4) s(-1) (at 1 mM O(2), pH 7.8) and 3. 7x10(3) s(-1), respectively. The first phase is attributed to binding of O(2) to heme a(3) and oxidation of both hemes forming the 'peroxy' intermediate. The second phase was associated with proton uptake from solution and it is attributed to formation of the 'oxo-ferryl' state, the final state in the absence of quinol. In the presence of bound caldariella quinol (QH(2)), heme a was re-reduced by QH(2) with a rate of 670 s(-1), followed by transfer of the fourth electron to the binuclear center with a rate of 50 s(-1). Thus, the results indicate that the quinol donates electrons to heme a, followed by intramolecular transfer to the binuclear center. Moreover, the overall electron and proton-transfer kinetics in the A. ambivalens quinol oxidase are the same as those in the E. coli ubiquinol oxidase, which indicates that in the A. ambivalens enzyme a different pathway is used for proton transfer to the binuclear center and/or other protonatable groups in an equivalent pathway are involved. Potential candidates in that pathway are two glutamates at positions (I-80) and (I-83) in the A. ambivalens enzyme (corresponding to Met(I-116) and Val(I-119), respectively, in E. coli cytochrome bo(3)).

Cytochrome c oxidase (CytcO) is a redox-driven proton pump in the respiratory chain of mitochondria and many aerobic bacteria. The results from several studies have shown that zinc ions interfere with both the uptake and release of protons, presumably by binding near the orifice of the proton entrance and exit pathways. To elucidate the effect of Zn2+ binding on individual electron and proton-transfer reactions, in this study, we have investigated the reaction of the fully reduced R. sphaeroides CytcO with O2, both with enzyme in detergent solution and reconstituted in phospholipid vesicles, and, with and without, Zn2+. The results show that addition of Zn2+ at concentrations ofor = 250 microM to the outside of the vesicles did not alter the transition rates between intermediates PR (P3)--F3--O4. However, proton pumping was impaired specifically during the P3--F3, but not during the F3--O4 transition at Zn2+ concentrations ofor = 25 microM. Furthermore, proton pumping during the P3--F3 transition was typically impaired with the "as isolated" CytcO, which was found to contain Zn2+ ions at microM concentration. As has already been shown, Zn2+ was also found to obstruct proton uptake during the P3--F3 transition, presumably by binding to a site near the orifice of the D-pathway. In this work we found a KI of approximately 1 microM for this binding site. In conclusion, the results show that Zn2+ ions bind on both sides of CytcO and that binding of Zn2+ at the proton output side selectively impairs proton release during the P3--F3 transition.

The cytochrome c and ubiquinol oxidases discussed in this article are membrane-bound redox-driven proton pumps which couple an electron current to a proton current across the membrane. This coupling requires a control of the thermodynamics and/or rates of internal electron- and proton-transfer reactions (termed 'gating'). Therefore, to understand the structure-function relation of these proton pumps, individual electron- and proton-transfer reactions must be investigated. We have undertaken such studies by using a combination of site-directed mutagenesis and spectroscopic techniques. The results show that proton uptake/release upon reduction/oxidation of heme a3 takes place on a ms-time scale through the K-pathway (including Thr(I-359) and Lys(I-362)), but not through the D-pathway (including Asp(I-132) and Glu(I-286)). During reaction of the reduced enzyme with O2, both substrate and pumped protons are taken up through the D-pathway (but not through the K-pathway) in a biphasic process with time constants of 100 microseconds and 1 ms. Thus, the original assignment of the role of the D-pathway (used only for pumped protons) must be revised. Dynamic studies of proton uptake to the enzyme surface show that on the proton-input side, the surface carries a proton-collecting antenna made of carboxylate and histidine residues which enable the enzyme to pick up protons with a rate compatible to the enzyme turnover rate. These results are consistent with the three-dimensional cytochrome c oxidase structure which shows that the entry point to the D-pathway (but not to the K-pathway) is surrounded by a network of histidine residues within a negative electrostatic potential.

Cytochrome oxidase is one of the functionally most intriguing redox-driven proton pumps. During the last decade our increased understanding of the system has greatly benefited from theoretical calculations and modeling in the framework of three-dimensional structures of cytochrome c oxidases from different species. Because these studies are based on results from experiments, it is important that any ambiguities in the conclusions extracted from these experiments are discussed and elucidated. In a recent study Szundi et al. (Szundi et al. Biochemistry 2012, 51, 9302) investigated the reaction of the reduced Rhodobacter sphaeroides cytochrome c oxidase with O2 and arrived at conclusions different from those derived from earlier investigations. In this short communication we compare these very recent data to those obtained from earlier studies and discuss the origin of the differences.

Heme-copper oxidases (HCuOs) catalyse the reduction of oxygen, using the liberated free energy to maintain a proton-motive force across the membrane. In the mitochondrial-like A-type HCuOs, binding of heavy metal ions at the surface of the protein inhibits proton transfer. In bacterial C-type oxidases, the entry point to the proton pathway is on an accessory subunit unrelated to any subunit in A-type HCuOs. Despite this, we show here that heavy metal ions such as Zn2+ inhibit O2-reduction very similarly in C-type as in A-type HCuOs, and furthermore that the binding site shares the same Glu-His motif.