Ólöf Einarsdóttir

CO impedes superfast O2 binding in ba3 cytochrome oxidase from Thermus thermophilus

Kinetic studies of heme-copper terminal oxidases using the CO flow-flash method are potentially compromised by the fate of the photodissociated CO. In this time-resolved optical absorption study, we compared the kinetics of dioxygen reduction by ba3 cytochrome c oxidase from Thermus thermophilus in the absence and presence of CO using a photolabile O2-carrier. A novel double-laser excitation is introduced in which dioxygen is generated by photolyzing the O2-carrier with a 355 nm laser pulse and the fully reduced CO-bound ba3 simultaneously with a second 532-nm laser pulse. A kinetic analysis reveals a sequential mechanism in which O2 binding to heme a3 at 90 μM O2 occurs with lifetimes of 9.3 and 110 μs in the absence and presence of CO, respectively, followed by a faster cleavage of the dioxygen bond (4.8 μs), which generates the P intermediate with the concomitant oxidation of heme b. The second-order rate constant of 1 × 109 M-1 s-1 for O2 binding to ba3 in the absence of CO is 10 times greater than observed in the presence of CO as well as for the bovine heart enzyme. The O2 bond cleavage in ba3 of 4.8 μs is also approximately 10 times faster than in the bovine enzyme. These results suggest important structural differences between the accessibility of O2 to the active site in ba3 and the bovine enzyme, and they demonstrate that the photodissociated CO impedes access of dioxygen to the heme a3 site in ba3, making the CO flow-flash method inapplicable.

A New Approach for Studying Fast Biological Reactions Involving Nitric Oxide: Generation of NO Using Photolabile Ruthenium and Manganese NO Donors

Abstract Nitric oxide (NO) is recognized as one of the major players in various biochemical processes, including blood pressure, neurotransmission and immune responses. However, experimental studies involving NO are often limited by difficulties associated with the use of NO gas, including its toxicity and precise control over NO concentration. Moreover, the reactions of NO with biological molecules, which frequently occur on time scales of microseconds or faster, are limited by the millisecond time scale of conventional stopped-flow techniques. Here we present a new approach for studying rapid biological reactions involving NO. The method is based on designed ruthenium and manganese nitrosyls, [Ru(PaPy3)(NO)](BF4)2 and [Mn(PaPy3)(NO)](ClO4) (PaPy3H = N,N–bis(2-pyridylmethyl)amine-N-ethyl-2-pyridine-2-carboxamide), which upon photolysis produce NO on a fast time scale. The kinetics of the binding of the photogenerated NO to reduced cytochrome c oxidase (CcO) and myoglobin (Mb) was investigated using time-resolved optical absorption spectroscopy. The NO was found to bind to reduced CcO with an apparent lifetime of 77 μs using the [Mn(PaPy3)(NO)]+ complex; the corresponding rate is 10–20 times faster than can be detected by conventional stopped-flow methods. Second-order rate constants of ∼1 × 108 M−1 s−1 and ∼3 × 107 M−1 s−1 were determined for NO binding to reduced CcO and Mb, respectively. The generation of NO by photolysis of these complexes circumvents the rate limitation of stopped-flow techniques and offers a novel alternative to study other fast biological reactions involving NO.

Time-resolved magnetic circular dichroism spectroscopy of photolyzed carbonmonoxy cytochrome c oxidase (cytochrome aa3).

Nanosecond time-resolved magnetic circular dichroism (TRMCD) and time-resolved natural circular dichroism (TRCD) measurements of photolysis products of the CO complex of eukaryotic cytochrome c oxidase (CcO-CO) are presented. TRMCD spectra obtained at 100 ns and 10 microseconds after photolysis are diagnostic of pentacoordinate cytochrome a3Fe2+, as would be expected for simple photodissociation. Other time-resolved spectroscopies (UV-visible and resonance Raman), however, show evidence for unusual Fea3(2+) coordination after CO photolysis (Woodruff, W. H., O. Einarsdóttir, R. B. Dyer, K. A. Bagley, G. Palmer, S. J. Atherton, R. A. Goldbeck, T. D. Dawes, and D. S. Kliger. 1991. Proc. Nat. Acad. Sci. U.S.A. 88:2588-2592). Furthermore, time-resolved IR experiments have shown that photodissociated CO binds to CuB+ prior to recombining with Fea3(2+) (Dyer, R. B., O. Einarsdóttir, P. M. Killough, J. J. López-Garriga, and W. H. Woodruff. 1989. J. Am. Chem. Soc. 111:7657-7659). A model of the CcO-CO photolysis cycle which is consistent with all of the spectroscopic results is presented. A novel feature of this model is the coordination of a ligand endogenous to the protein to the Fe axial site vacated by the photolyzed CO and the simultaneous breaking of the Fe-imidazole(histidine) bond.

Kinetic studies of the reactions of O2 and NO with reduced Thermus thermophilus ba3 and bovine aa3 using photolabile carriers

The reactions of molecular oxygen (O(2)) and nitric oxide (NO) with reduced Thermus thermophilus (Tt) ba(3) and bovine heart aa(3) were investigated by time-resolved optical absorption spectroscopy to establish possible relationships between the structural diversity of these enzymes and their reaction dynamics. To determine whether the photodissociated carbon monoxide (CO) in the CO flow-flash experiment affects the ligand binding dynamics, we monitored the reactions in the absence and presence of CO using photolabile O(2) and NO complexes. The binding of O(2)/NO to reduced ba(3) in the absence of CO occurs with a second-order rate constant of 1×10(9)M(-1)s(-1). This rate is 10-times faster than for the mammalian enzyme, and which is attributed to structural differences in the ligand channels of the two enzymes. Moreover, the O(2)/NO binding in ba(3) is 10-times slower in the presence of the photodissociated CO while the rates are the same for the bovine enzyme. This indicates that the photodissociated CO directly or indirectly impedes O(2) and NO access to the active site in Tt ba(3), and that traditional CO flow-flash experiments do not accurately reflect the O(2) and NO binding kinetics in ba(3). We suggest that in ba(3) the binding of O(2) (NO) to heme a(3)(2+) causes rapid dissociation of CO from Cu(B)(+) through steric or electronic effects or, alternatively, that the photodissociated CO does not bind to Cu(B)(+). These findings indicate that structural differences between Tt ba(3) and the bovine aa(3) enzyme are tightly linked to mechanistic differences in the functions of these enzymes. This article is part of a Special Issue entitled: Respiratory Oxidases.

PM and PR forms of cytochrome c oxidase have different spectral properties

The reaction between bovine heart cytochrome c oxidase and dioxygen was monitored at room temperature in the visible and Soret regions following photolysis of the mixed-valence CO-bound enzyme. Time-resolved optical absorption difference spectra were collected between 50 ns and 1.7 ms by a gated multichannel analyzer. Singular value decomposition and global exponential fitting resolved three processes with apparent lifetimes of 2.2+/-0.5, 17+/-4 and 160+/-30 micros. The spectra of the intermediates were extracted based on a sequential kinetic mechanism and compared to the corresponding intermediate spectra observed during the reaction of the fully reduced enzyme with dioxygen. The first process is associated with a conformational change at heme a(3) upon dissociation of CO from Cu(B)(+) and concomitant back-electron transfer from heme a(3) to heme a. This is followed by O(2) binding to heme a(3) forming compound A (A(M)), with a spectrum identical to that observed upon O(2) binding to heme a(3) in the fully reduced enzyme (A(R)). Intermediate A(M) decays into P(M), the spectrum of which is equivalent to that of the 607 nm form, generated upon addition of H(2)O(2) to the oxidized enzyme at alkaline pH values (P(H)). However, the spectrum of P(M) is significantly different from the corresponding intermediate observed upon the reaction of dioxygen with the fully reduced enzyme (P(R)). The spectral differences between P(M) and P(R) may arise from the different number of redox equivalents at the binuclear site, with a tyrosine radical in the P(M) state, and tyrosine or tyrosinate in P(R), or may be the consequence of a more complex reaction mechanism in the case of the fully reduced enzyme.

Time-Resolved Studies of the Excited-State Dynamics of meso-Tetra(hydroxylphenyl)chlorin in Solution

Abstract— Meso‐tetra(hydroxyphenyl)chlorin (m‐THPC) is a new photosensitizer developed for potential use in photodynamic therapy (PDT) for cancer treatment. In PDT, the accepted mechanism of tumor destruction involves the formation of excited singlet oxygen via intermolecular energy transfer from the excited triplet‐state dye to the ground triplet‐state oxygen. Femtosecond transient absorption measurements are reported here for the excited singlet state dynamics of m‐THPC in solution. The observed early time kinetics were best fit using a triple exponential function with time constants of 350 fs, 80 ps and 3.3 ns. The fastest decay (350 fs) was attributed to either internal conversion from S2 to S1 or vibrational relaxation in S2. Multichannel time‐resolved absorption and emission spectroscopies were also used to characterize the excited singlet and triplet states of the dye on nanosecond to microsecond time scales at varying concentrations of oxygen. The nanosecond time‐resolved absorption data were fit with a double exponential with time constants of 14 ns and 250 ns in ambient air, corresponding to lifetimes of the S1 and T1 states, respectively. The decay of the T1 state varied linearly with oxygen concentration, from which the intrinsic decay rate constant, ki, of 1.5 × 106 s−1 and the bimolecular collisional quenching constant, kc, of 1.7 × 109M−1 s−1 were determined. The lifetime of the S1 state of 10 ns was confirmed by fluorescence measurements. It was found to be independent of oxygen concentration and longer than lifetimes of other photosensitizers.

The pathway of O2 to the active site in heme–copper oxidases

The route of O₂to and from the high-spin heme in heme-copper oxidases has generally been believed to emulate that of carbon monoxide (CO). Time-resolved and stationary infrared experiments in our laboratories of the fully reduced CO-bound enzymes, as well as transient optical absorption saturation kinetics studies as a function of CO pressure, have provided strong support for CO binding to CuB⁺ on the pathway to and from the high-spin heme. The presence of CO on CuB⁺ suggests that O₂binding may be compromised in CO flow-flash experiments. Time-resolved optical absorption studies show that the rate of O₂and NO binding in the bovine enzyme (1 × 10⁸M⁻¹s⁻¹) is unaffected by the presence of CO, which is consistent with the rapid dissociation (t½ = 1.5μs) of CO from CuB⁺. In contrast, in Thermus thermophilus (Tt) cytochrome ba3 the O₂and NO binding to heme a3 slows by an order of magnitude in the presence of CO (from 1 × 10⁹ to 1 × 10⁸M⁻¹s⁻¹), but is still considerably faster (~10μs at 1atm O₂) than the CO off-rate from CuB in the absence of O₂(milliseconds). These results show that traditional CO flow-flash experiments do not give accurate results for the physiological binding of O₂and NO in Tt ba3, namely, in the absence of CO. They also raise the question whether in CO flow-flash experiments on Tt ba3 the presence of CO on CuB⁺ impedes the binding of O₂to CuB⁺ or, if O₂does not bind to CuB⁺ prior to heme a3, whether the CuB⁺-CO complex sterically restricts access of O₂to the heme. Both possibilities are discussed, and we argue that O₂binds directly to heme a3 in Tt ba3, causing CO to dissociate from CuB⁺ in a concerted manner through steric and/or electronic effects. This would allow CuB⁺ to function as an electron donor during the fast (5μs) breaking of the OO bond. These results suggest that the binding of CO to CuB⁺ on the path to and from heme a3 may not be applicable to O₂and NO in all heme-copper oxidases. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.

Spectral identification of intermediates generated during the reaction of dioxygen with the wild-type and EQ(I-286) mutant of rhodobacter sphaeroides cytochrome c oxidase

Cytochrome c oxidase from Rhodobacter sphaeroides is frequently used to model the more complex mitochondrial enzyme. The O(2) reduction in both enzymes is generally described by a unidirectional mechanism involving the sequential formation of the ferrous-oxy complex (compound A), the P(R) state, the oxyferryl F form, and the oxidized state. In this study we investigated the reaction of dioxygen with the wild-type reduced R. sphaeroides cytochrome oxidase and the EQ(I-286) mutant using the CO flow-flash technique. Singular value decomposition and multiexponential fitting of the time-resolved optical absorption difference spectra showed that three apparent lifetimes, 18 μs, 53 μs, and 1.3 ms, are sufficient to fit the kinetics of the O(2) reaction of the wild-type enzyme. A comparison of the experimental intermediate spectra with the corresponding intermediate spectra of the bovine enzyme revealed that P(R) is not present in the reaction mechanism of the wild-type R. sphaeroides aa(3). Transient absorbance changes at 440 and 610 nm support this conclusion. For the EQ(I-286) mutant, in which a key glutamic residue in the D proton pathway is replaced by glutamine, two lifetimes, 16 and 108 μs, were observed. A spectral analysis of the intermediates shows that the O(2) reaction in the EQ(I-286) mutant terminates at the P(R) state, with 70% of heme a becoming oxidized. These results indicate significant differences in the kinetics of O(2) reduction between the bovine and wild-type R. sphaeroides aa(3) oxidases, which may arise from differences in the relative rates of internal electron and proton movements in the two enzymes.

Photoreactions of Cytochrome c Oxidase

Abstract The photoreduction of oxidized bovine heart cytochrome c oxidase (CcO) by visible and UV radiation was investigated in the absence and presence of external reagents. In the former case, the quantum yields for direct photoreduction of heme A (heme a + heme a3) were 2.6 ± 0.5 × 10−3, 4 ± 1 × 10−4, and 4 ± 2 × 10−6 with pulsed laser irradiation at 266, 355 and 532 nm, respectively. Within experimental uncertainty, the quantum yields did not depend on pulse energy, implying that the mechanism is monophotonic. Irradiation with 355 nm light resulted in spectral changes similar to those produced independently by reduction with dithionite, whereby the low-spin heme a and CuA are reduced first. Extended illumination at 355 and 532 nm yielded substantial amounts of reduced heme a3. Heme decomposition was noted with 266 nm light. In the presence of formate and cyanide ions, which bind at the binuclear heme a3/copper center in CcO, irradiation at 355 nm caused selective reduction of only the low-spin heme a and CuA. The addition of ferrioxalate ion dramatically increased the efficiency of cytochrome c oxidase photoreduction. The quantum efficiency for heme A reduction was found to be near unity, significantly greater than for other known methods of photoreduction. The active reductant is most likely ferrous iron, and its reduction of the enzyme is thermodynamically driven by the reformation of ferrioxalate in the presence of excess oxalate ion. Other metalloenzymes with redox potentials similar to those of cytochrome c oxidase should be amenable to indirect photoreduction by this method.

Evidence for a band III analogue in the near-infrared absorption spectra of cytochrome c oxidase.

Ground state near-infrared absorption spectra of fully reduced unliganded and fully reduced CO (a2+ CuA+ a3(2+)-CO CuB+) cytochrome c oxidase were investigated. Flash-photolysis time-resolved absorption difference spectra of the mixed-valence (a3+ CuA2+ a3(2+)-CO CuB+) and the fully reduced CO complexes were also studied. A band near 785 nm (epsilon approximately 50 M-1cm-1) was observed in the fully reduced unliganded enzyme and the CO photoproducts. The time-resolved 785 nm band disappeared on the same timescale (t1/2 approximately 7 ms) as CO recombined with cytochrome a3(2+). This band, which is attributed to the unliganded five coordinate ferrous cytochrome a3(2+), has some characteristics of band III in deoxy-hemoglobin and deoxy-myoglobin. A second band was observed at approximately 710 nm (epsilon approximately 80 M-1cm-1) in the fully reduced unliganded and the fully reduced CO complexes. This band, which we assign to the low spin ferrous cytochrome a, appears to be affected by the ligation state at the cytochrome a3(2+) site.