The Reactions of O2 and NO with Mixed-Valence ba3 Cytochrome c Oxidase from Thermus thermophilus.

Earlier CO flow-flash experiments on the fully reduced Thermus thermophilus ba3 (Tt ba3) cytochrome oxidase revealed that O2 binding was slowed down by a factor of 10 in the presence of CO (Szundi et al., 2010, PNAS 107, 21010-21015). The goal of the current study is to explore whether the long apparent lifetime (∼50 ms) of the CuB+-CO complex generated upon photolysis of the CO-bound mixed-valence Tt ba3 (Koutsoupakis et al., 2019, Acc. Chem. Res. 52, 1380-1390) affects O2 and NO binding and the ability of CuB to act as an electron donor during O-O bond splitting. The CO recombination, NO binding, and the reaction of mixed-valence Tt ba3 with O2 were investigated by time-resolved optical absorption spectroscopy using the CO flow-flash approach and photolabile O2 and NO carriers. No electron backflow was detected after photolysis of the mixed-valence CO-bound Tt ba3. The rate of O2 and NO binding was two times slower than in the fully reduced enzyme in the presence of CO and 20 times slower than in the absence of CO. The purported long-lived CuB+-CO complex did not prevent O-O bond splitting and the resulting PM formation, which was significantly faster (5-10 times) than in the bovine heart enzyme. We propose that O2 binding to heme a3 in Tt ba3 causes CO to dissociate from CuB+ in a concerted manner through steric and/or electronic effects, thus allowing CuB+ to act as an electron donor in the mixed-valence enzyme. The significantly faster O2 binding and O-O bond cleavage in Tt ba3 compared to analogous steps in the aa3 oxidases could reflect evolutionary adaptation of the enzyme to the microaerobic conditions of the T. thermophilus HB8 species.

Role of the Conserved Valine 236 in Access of Ligands to the Active Site of Thermus thermophilus ba3 Cytochrome Oxidase.

Knowledge of the role of conserved residues in the ligand channel of heme-copper oxidases is critical for understanding how the protein scaffold modulates the function of these enzymes. In this study, we investigated the role of the conserved valine 236 in the ligand channel of ba3 cytochrome c oxidase from Thermus thermophilus by mutating the residue to a more polar (V236T), smaller (V236A), or larger (V236I, V236N, V236L, V236M, and V236F) residue. The crystal structures of the mutants were determined, and the effects of the mutations on the rates of CO, O2, and NO binding were investigated. O2 reduction and NO binding were unaffected in V236T, while the oxidation of heme b during O-O bond cleavage was not detected in V236A. The V236A results are attributed to a decrease in the rate of electron transfer between heme b and heme a3 during O-O bond cleavage in V236A, followed by faster re-reduction of heme b by CuA. This interpretation is supported by classical molecular dynamics simulations of diffusion of O2 to the active site in V236A that indicated a larger distance between the two hemes compared to that in the wild type and increased contact of heme a3 with water and weakened interactions with residues R444 and R445. As the size of the mutant side chain increased and protruded more into the ligand cavity, the rates of ligand binding decreased correspondingly. These results demonstrate the importance of V236 in facilitating access of ligands to the active site in T. thermophilus ba3.

The CO Photodissociation and Recombination Dynamics of the W172Y/F282T Ligand Channel Mutant of Rhodobacter sphaeroides aa3 Cytochrome c Oxidase.

In the ligand channel of the cytochrome c oxidase from Rhodobacter sphaeroides (Rs aa3 ) W172 and F282 have been proposed to generate a constriction that may slow ligand access to and from the active site. To explore this issue, the tryptophan and phenylalanine residues in Rs aa3 were mutated to the less bulky tyrosine and threonine residues, respectively, which occupy these sites in Thermus thermophilus (Tt) ba3 cytochrome oxidase. The CO photolysis and recombination dynamics of the reduced wild-type Rs aa3 and the W172Y/F282T mutant were investigated using time-resolved optical absorption spectroscopy. The spectral changes associated with the multiple processes are attributed to different conformers. The major CO recombination process (44 µs) in the W172Y/F282T mutant is ~500 times faster than the predominant CO recombination process in the wild-type enzyme (~23 ms). Classical dynamic simulations of the wild-type enzyme and double mutant showed significant structural changes at the active site in the mutant, including movement of the heme a3 ring-D propionate toward CuB and reduced binuclear center cavity volume. These structural changes effectively close the ligand exit pathway from the binuclear center, providing a basis for the faster CO recombination in the double mutant.

The pathway of O2to 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.

Kinetics and intermediates of the reaction of fully reduced Escherichia coli bo3 ubiquinol oxidase with O2.

Cytochrome bo3 ubiquinol oxidase from Escherichia coli catalyzes the reduction of O2 to water by ubiquinol. The reaction mechanism and the role of ubiquinol continue to be a subject of discussion. In this study, we report a detailed kinetic scheme of the reaction of cytochrome bo3 with O2 with steps specific to ubiquinol. The reaction was investigated using the CO flow-flash method, and time-resolved optical absorption difference spectra were collected from 1 µs to 20 ms after photolysis. Singular value decomposition-based global exponential fitting resolved five apparent lifetimes, 22 µs, 30 µs, 42 µs, 470 µs, and 2.0 ms. The reaction mechanism was derived by an algebraic kinetic analysis method using frequency-shifted spectra of known bovine states to identify the bo3 intermediates. It shows 42 µs O2 binding (3.8 × 10(7) M(-1) s(-1)), producing compound A, followed by faster (22 µs) heme b oxidation, yielding a mixture of PR and F, and rapid heme b rereduction by ubiquinol (30 µs), producing the F intermediate and semiquinone. In the 470 µs step, the o3 F state is converted into the o3(3+) oxidized state, presumably by semiquinone/ubiquinol, without the concomitant oxidation of heme b. The final 2 ms step shows heme b reoxidation and the partial rereduction of the binuclear center and, following O2 binding, the formation of a mixture of P and F during a second turnover cycle. The results show that ubiquinol/semiquinone plays a complex role in the mechanism of O2 reduction by bo3, displaying kinetic steps that have no analogy in the CuA-containing heme-copper oxidases.

Conserved glycine 232 in the ligand channel of ba3 cytochrome oxidase from Thermus thermophilus.

Knowing how the protein environment modulates ligand pathways and redox centers in the respiratory heme-copper oxidases is fundamental for understanding the relationship between the structure and function of these enzymes. In this study, we investigated the reactions of O2 and NO with the fully reduced G232V mutant of ba3 cytochrome c oxidase from Thermus thermophilus (Tt ba3) in which a conserved glycine residue in the O2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa3 cytochrome c oxidase suggested that the valine completely blocked the access of O2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617-11621]. Using photolabile O2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O2 and NO binding are not significantly affected in the Tt ba3 G232V mutant. Classical molecular dynamics simulations of diffusion of O2 to the active site in the wild-type enzyme and G232V mutant show that the insertion of the larger valine residue in place of the glycine appears to open up other O2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.

Ligand access to the active site in Thermus thermophilus ba(3) and bovine heart aa(3) cytochrome oxidases.

Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates the functions of these enzymes. Using photolabile NO and O(2) carriers, we recently found that NO and O(2) binding in Thermus thermophilus (Tt) ba(3) is ~10 times faster than in the bovine enzyme, indicating that inherent structural differences affect ligand access in these enzymes. Using X-ray crystallography, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in wild-type Tt ba(3) and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O(2) channel of Tt ba(3) are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the aa(3) enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type ba(3) and the T231F mutant. The results show that the Tt ba(3) Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic "way station", which may further slow ligand access to the active site. Classical simulations of diffusion of Xe to the active sites in ba(3) and bovine aa(3) show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba(3) Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.

Biophysical Characterization of α-Synuclein and Rotenone Interaction.

Previous studies revealed that pesticides interact with α-synuclein and accelerate the rate of fibrillation. These results are consistent with the prevailing hypothesis that the direct interaction of α-synuclein with pesticides is one of many suspected factors leading to α-synuclein fibrillation and ultimately to Parkinson's disease. In this study, the biophysical properties and fibrillation kinetics of α-synuclein in the presence of rotenone were investigated and, more specifically, the effects of rotenone on the early-stage misfolded forms of α-synuclein were considered. The thioflavine T (ThT) fluorescence assay studies provide evidence that early-phase misfolded α-synuclein forms are affected by rotenone and that the fibrillation process is accelerated. Further characterization by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) shows that rotenone increases the amount of ordered secondary structure in this intrinsically disordered protein. Morphological characterization by transmission electron microscopy (TEM) and atomic force microscopy (AFM) provide visualization of the differences in the aggregated α-synuclein species developing during the early kinetics of the fibrillation process in the absence and presence of rotenone. We believe that these data provide useful information for a better understanding of the molecular basis of rotenone-induced misfolding and aggregation of α-synuclein.

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.

Kinetic studies of the reactions of O(2) and NO with reduced Thermus thermophilus ba(3) and bovine aa(3) 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.

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 ba(3) cytochrome c oxidase from Thermus thermophilus in the absence and presence of CO using a photolabile O(2)-carrier. A novel double-laser excitation is introduced in which dioxygen is generated by photolyzing the O(2)-carrier with a 355 nm laser pulse and the fully reduced CO-bound ba(3) simultaneously with a second 532-nm laser pulse. A kinetic analysis reveals a sequential mechanism in which O(2) binding to heme a(3) at 90 µM O(2) 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 × 10(9) M(-1) s(-1) for O(2) binding to ba(3) 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 O(2) bond cleavage in ba(3) 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 O(2) to the active site in ba(3) and the bovine enzyme, and they demonstrate that the photodissociated CO impedes access of dioxygen to the heme a(3) site in ba(3), making the CO flow-flash method inapplicable.

Solvent effects on the physicochemical properties of the cross-linked histidine-tyrosine ligand of cytochrome c oxidase.

Density functional theory was used to explore the effects of aqueous solvation on the structure, vibrational frequencies, and the electronic absorption spectrum of 2-(4-methylimidazol-1-yl)-phenol (Me-ImPhOH), a chemical analogue of the cross-linked histidine-tyrosine Cu(B) ligand of cytochrome c oxidase. In addition, the phenolic-OH pK(a), the anodic redox potential for the biring radical/anion couple, and the phenolic-OH bond dissociation energy were calculated relative to phenol using a series of isodesmic reactions. In the gas phase, the imidazole moiety stabilizes the biring anion for all the models and greatly decreases the phenolic-OH pK(a) relative to phenol. Moreover, the conductor-like polarizable continuum model (C-PCM)-water-solvated reactions predict Delta pK(a) values that are five times smaller than the gas-phase reactions, in agreement with the proposed role of the cross-linked histidine-tyrosine as a proton donor in the enzyme. For the neutral biring radical solvation models, the imidazole moiety induces a high degree of asymmetry into the phenol ring when compared to unmodified phenoxyl radical. The biring radical pi-bonds of the imidazole ring are more localized when compared to unmodified 1-methylimidazole and Me-ImPhOH solvation models, suggesting reduced aromaticity for all biring radical solvation models. The C-PCM-water-solvated reactions predict relative biring radical reduction potentials that are an order of magnitude smaller than the gas-phase reactions. The biring O-H bond is weakened relative to phenol by less than 4 kcal/mol for all the reactions studied, suggesting that the imidazole moiety does not facilitate H-atom abstraction in the enzyme. Together, these results demonstrate the sensitive nature of the proton and electron donating ability of the histidine-tyrosine cross-linked ligand in cytochrome c oxidase and suggest that for quantitative predictions of reaction energies and thermodynamic properties, models of this ligand should take care to account for changes in environment and, more specifically, hydrogen bonding interactions.

Synthesis of a cyclic pentapeptide mimic of the active site His-Tyr cofactor of cytochrome c oxidase.

Arylboronic acid based technology provides a mild, regioselective, and nontoxic N-arylation procedure for accessing the unusual N-arylated side chain histidine found in the active site of cytochrome c oxidase (CcO). The N-arylated histidine is elaborated to the complete cytochrome c oxidase cyclic pentapeptide cofactor. Molecular modeling of the cofactor provides insight into the dynamic character of the N-aryl bond.

A spectroscopic investigation of a tridentate Cu-complex mimicking the tyrosine-histidine cross-link of cytochrome C oxidase.

Heme-copper oxidases have a crucial role in the energy transduction mechanism, catalyzing the reduction of dioxygen to water. The reduction of dioxygen takes place at the binuclear center, which contains heme a3 and CuB. The X-ray crystal structures have revealed that the C6' of tyrosine 244 (bovine heart numbering) is cross-linked to a nitrogen of histidine 240, a ligand to CuB. The role of the cross-linked tyrosine at the active site still remains unclear. In order to provide insight into the function of the cross-linked tyrosine, we have investigated the spectroscopic and electrochemical properties of chemical analogues of the CuB-His-Tyr site. The analogues, a tridentate histidine-phenol cross-linked ether ligand and the corresponding Cu-containing complex, were previously synthesized in our laboratory (White, K.; et al. Chem. Commun. 2007, 3252-3254). Spectrophotometric titrations of the ligand and the Cu-complex indicate a pKa of the phenolic proton of 8.8 and 7.7, respectively. These results are consistent with the cross-linked tyrosine playing a proton delivery role at the cytochrome c oxidase active site. The presence of the phenoxyl radical was investigated at low temperature using electron paramagnetic resonance (EPR) and Fourier transform infrared (FT-IR) difference spectroscopy. UV photolysis of the ligand, without bound copper, generated a narrow g=2.0047 signal, attributed to the phenoxyl radial. EPR spectra recorded before and after UV photolysis of the Cu-complex showed a g=2 signal characteristic of oxidized copper, suggesting that the copper is not spin-coupled to the phenoxyl radical. An EPR signal from the phenoxyl radical was not observed in the Cu-complex, either due to spin relaxation of the two unpaired electrons or to masking of the narrow phenoxyl radical signal by the strong copper contribution. Stable isotope (13C) labeling of the phenol ring (C1') Cu-complex, combined with photoinduced difference FT-IR spectroscopy, revealed bands at 1485 and 1483 cm(-1) in the 12C-minus-13C-isotope-edited spectra of the ligand and Cu-complex, respectively. These bands are attributed to the radical v7a stretching frequency and are shifted to 1468 and 1472 cm(-1), respectively, with 13C1' labeling. These results show that a radical is generated in both the ligand and the Cu-complex and support the unambiguous assignment of a vibrational band to the phenoxyl radical v7a stretching mode. These data are discussed with respect to a possible role of the cross-linked tyrosine radical in cytochrome c oxidase.

Solvent effects on the vibrational frequencies of the phenolate anion, the para-cresolate anion, and their radicals.

The effects of aqueous solvation on the structure and vibrational frequencies of phenol, para-cresol, and their respective radicals are calculated at the B3LYP/6-31+G(d,p) level of theory using the conductor-like polarizable continuum model (C-PCM) alone and in combination with an explicit water molecule H-bonded to the phenolic oxygen. Calculated vibrational frequencies are compared to experimental frequencies obtained in aqueous buffer at high pH. For all models, the C-PCM provides the best overall agreement between theory and experiment at a modest computational effort, as demonstrated by the lowest mean absolute deviations in the computed frequencies. In addition, the C-PCM provides anion Wilson mode 7a (18)O isotope shifts in excellent agreement with experiment and improves agreement between the computed and observed radical Wilson mode 7a (2)H isotope shift. On the basis of a quantitative comparison of the anion and radical normal modes by vibrational projection analysis and total energy decomposition, an alternative criterion for distinguishing the anion and radical Wilson modes 7a and 19a using the relative phasing of the carbon-oxygen and carbon-carbon bond stretches is presented.

Flash-photolysis of fully reduced and mixed-valence CO-bound Rhodobacter sphaeroides cytochrome c oxidase: heme spectral shifts.

Conformational changes, internal electron transfer, and CO rebinding processes in cytochrome c oxidase from Rhodobacter sphaeroides reduced to different degrees were investigated. The reactions were followed using a gated optical spectrometric multichannel analyzer. Light-induced difference spectra, recorded in the 350-700 nm region over the 100 ns to 1 s time interval, were analyzed by singular value decomposition and global exponential fitting. The photolyzed fully reduced enzyme showed two relaxations, approximately 1 and 190 mus, prior to the 20 ms CO rebinding process. Intramolecular electron transfer was monitored following photolysis of the mixed-valence CO-bound enzyme. The analysis revealed 1.1 micros, 2.4 micros, 31 micros, 68 ms, and 240 ms apparent lifetimes, the first three of which are attributed to electron transfer from heme a3 to heme a with contribution from a relaxation process at the heme a3 site. Spectral changes associated with the microsecond processes are consistent with 75% electron transfer from heme a3 to heme a. A comparison of the experimental spectra and model difference spectra for the intramolecular electron transfer indicated approximately 3 nm blue shift in the absolute spectra of both the oxidized heme a3 and reduced heme a generated in the process. The 68 and 240 ms lifetimes are due to CO recombination to heme a3 and are attributed to the presence of two conformers, the slower rate corresponding to the conformer in higher abundance. The dependency of the apparent rate of CO rebinding on the intensity of the probe beam in single-wavelength experiments is explained.

Synthesis and structural characterization of cross-linked histidine-phenol Cu(ii) complexes as cytochrome c oxidase active site models.

Tridentate cross-linked histidine-phenol Cu(ii) ether and ester complexes, chemical analogs of the active site of several heme-copper oxidases, have been synthesized and crystallized.

Photoreactions of cytochrome C oxidase.

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 a(3)) were 2.6 +/- 0.5 x 10(-3), 4 +/- 1 x 10(-4), and 4 +/- 2 x 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 Cu(A) are reduced first. Extended illumination at 355 and 532 nm yielded substantial amounts of reduced heme a(3). Heme decomposition was noted with 266 nm light. In the presence of formate and cyanide ions, which bind at the binuclear heme a(3)/copper center in CcO, irradiation at 355 nm caused selective reduction of only the low-spin heme a and Cu(A). 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.

A new approach for studying fast biological reactions involving nitric oxide: generation of NO using photolabile ruthenium and manganese NO donors.

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 micros 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 approximately 1 x 10(8) M(-1) s(-1) and approximately 3 x 10(7) 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.