After the transfer of the third electron to the oxygen reduction site at low temperatures, two reactive oxygen intermediates are successively formed at the oxygen reduction site. By studying the reoxidation of partially reduced cytochrome c oxidase at low temperatures, we also demonstrate that the rate of electron transfer within the oxidase depends on the nature of the trapped dioxygen intermediate at the site of oxygen reduction. Finally, we took advantage of the unique reaction between another three-electron-reduced intermediate and carbon monoxide to probe the coordination sphere of the copper center at the oxygen reduction reactive site of cytochrome c oxidase.
Since the cytochrome c binding site(s) is located on the cytosolic side of the inner mitochondrial membrane ( 22 ), it is likely that Fea is also located close to the cytosolic side of the membrane. Therefore, it has been hypothesized that CuA is also located in a subunit on the cytosolic side of the membrane (32). It is generally assumed that this part of the protein complex is embedded in the bilayer membrane, closer to the matrix.
The distribution function (g(k)) is then determined by taking the mverse Laplace transform (L - 1) of the power law function (Eqns. 8-9). First-order rate constants and corresponding temperatures for the formation of the CuA EPR signal. Intensity of the unusual Cua EPR signal during incubation of a reduced sample of cytochrome c oxidase in the presence of dioxygen at 181 K.
First-order rate constants and corresponding temperatures for the Cua EPR signal decay. Activation enthalpy distribution plot for the decay of the unusual Cua EPR signal at 203 K.
Scheme III
If Scheme III is correct, it must somehow account for the unusual EPR spectroscopic properties of the two reduced three-electron intermediates. Regarding the first intermediate, we need to explain why only a small part of the EPR spectrum of copper is observed and why this signal is very difficult to saturate compared to magnetically isolated copper ions. The resistance of the unusual Cua EPR signal to power saturation also results from weak superexchange coupling to the iron ion.
The presence of nearby high-lying states in the iron spin manifold, which are mixed with the copper spin states, will facilitate relaxation of the copper spin via the Orbach mechanism (31). Thus, all the properties of the unusual Cua signal are accounted for by assuming the structure presented in Scheme III and a fairly weak (1.1 or 3.5 cm-1, depending on the iron spin state) superexchange interaction between the copper and the to postulate iron. In the third step of the reaction, an additional fraction of Fea is oxidized.
This is not surprising given the changes that necessarily occur at the dioxygen reduction site during the reaction cycle. Since complete recovery of the normal CuA signal intensity is observed after incubation at the low temperatures, we have. These results suggest that a three-electron reduced intermediate is involved in the production of the magnetically isolated Cua EPR signal.
Alternative explanations for the appearance of the magnetically isolated EPR signals that do not involve re-reduction of this location must assume that the.
3 Signal
The position of the a-band in the optical spectrum of the 428/580 nm species is unusually blue-shifted relative to the optical spectrum of the resting state of cytochrome c oxidase (Fig. 4a). Incomplete formation of the 428/580 nm species may result if excess H O is added to the resting state of the enzyme (19), as all molecules are unlikely to be activated, and if excess H O is added to the pulsed enzyme in the presence of catalase in the solution (20). After the reduction and reoxidation of the sample with excess H O (3.5 mM), the optical spectrum of the 428/580 nm species was obtained (b, solid trace).
The spectral features in the final optical spectrum correspond to the conversion from the 4281580 nm species to the resting state of the enzyme. It is noteworthy that the optical spectrum of the 428-580 nm species resembles the optical spectrum of the cyanide-inhibited enzyme (Fe~~1-CN CuM). 7). Optical spectra of the cyanide-inhibited enzyme (dashed line) and the 428/580 nm form of the enzyme (solid trace) are shown in panel a.
Cua, is expected to lead to the formation of the rhombic Cua EPR signal, similar to the reaction observed at low temperatures (Chapter II). After reoxidation of the reduced sample (spectrum not shown) with excess H 2 O 2 , the optical spectrum in Fig. The absorption spectrum of the 428/580 nm species, recorded 90 sec after the addition of H 2 O 2 , is shown in panel a (solid trace). .
Absorption and EPR spectra of the 428/580 nm species before and after CO addition.
OPTICAL
0 CuB
One reaction pathway was proposed to be independent of carbon monoxide pressure and involved the initial formation of a copper(I)-CO complex. The lack of an EPR signal from the binuclear dioxygen reduction site of the 428/580 nm species is puzzling, although it does not rule out that the site is composed of a paramagnetic species. Mossbauer spectroscopy was used to probe the EPR-silent phosphate-inhibited complex of the purple acid phosphatase enzyme uteroferrin (38).
Fe118/copper Cua pair cytochrome c oxidase, even if the binuclear center of the uteroferrin phosphate complex has a ground state of S = 1/2, it is conceivable that the species could still be EPR silent if the magnitude of the superexchange coupling is comparable to the zero-field splitting of the iron centers. An alternative proposal for the reactive species at the dioxygen reduction site in the 428/580 nm species could be considered. Typically, these rather unusual intermediates are formed by the two-electron oxidation of the native iron peroxidases (such as horseradish peroxidase (HRP) or Japanese radish) and catalase (CAT) by hydrogen peroxide (equation 2), yielding an intermediate. diate which is formally Fe(V) (2, 39) (these high-quality enzymatic intermediates are referred to as Compounds I).
Studies on model compounds have elucidated some of the factors governing the formation of a stable, reversibly oxidized/reduced porphyrin (40). Heme A contains an isoprenoid chain and a formyl group at the 2nd and 8th positions of the porphyrin ring, in place of the methyl and vinyl groups found on protoporphyrin IX (Fig. 17). Because of the strong electron-withdrawing effect of the formyl group, Chan and co-workers (41) concluded that heme A is probably resistant to reversible one-electron oxidation, making it quite unlikely that a porphyrin 1r radical cation of heme A is formed after the reaction of cytochrome c oxidase with hydrogen peroxide.
This model requires a large shift in the reduction potential of Cua during reaction with CO, from a high-potential form capable of resisting oxidation by the ferryl porphyrin radical cation 1r, to a low-potential form, e which exhibits a lower reduction potential than ferric Fe01 • A copper complex exhibiting such a difference in reduction potentials seems unlikely.
CHz I
In the complete absence of H 2 O 2 , the pulsatile enzyme is produced after reoxidation of the reduced enzyme by dithionite (27). In the absence of reducing equivalents, this reactive form of the enzyme eventually converts to the resting state of the enzyme (15). ii{2). There has been considerable speculation regarding the spin and redox states of Feaa in this state of the enzyme.
The frequency of the oxidation state marker band, v 4 , is believed to be inversely proportional to the electron population in the empty porphyrin 1r • molecular orbitals (8,10,11). The normal mode that is sensitive to the oxidation state of the metal is shown in Figure 1. The normal mode (v2) that is sensitive to the spin state of the iron is shown in Figure 1.
The preparation of the 428/580 nm species was accomplished by adding excess H 202 (4-6 mM) to samples of the fully reduced enzyme. Due to the intense absorption of the samples, transmission filters (16-50 %) were used to attenuate the reference beam. In order to avoid prolonged irradiation of only one surface of the sample (contained in the EPR tube), the sample was continuously rotated during the irradiation period (45-200 min).
Resonance Raman spectra of quiescent cytochrome c oxidase in the liquid (273 K) and solid (153 K) states are shown in Figure 2a and 2b.
Vibrational modes from both heme centers are observed in the spectrum of the resting enzyme; however several bands of Fea. There were also some changes evident in the spectrum of the frozen sample relative to the spectrum of the liquid sample: (i) The band centered at 1648 cm-1, which is a composite of several modes, is more symmetrical and narrower than in the corresponding spectrum of the resting enzyme in the liquid state, (ii) the 1675 cm-1 peak, assigned to the formyl group associated with Fe118, has increased in intensity, and (iii) there is more pronounced intensity at 1589 cm -1, which is indicative of increased scattering of a low-spin ferric Fe118 species (Table II). Our high-frequency resonance Raman spectrum of the pulsed form of cytochrome c oxidase is shown in Figure 4a.
Stoichiometrically reduced cytochrome c oxidase was reoxidized with 1 atm O:z to produce the pulse form of the enzyme (a). A follow-up sample of the peroxide adduct was prepared by adding approximately one equivalent of hydrogen peroxide to the pulsating enzyme (Figure 4c). The most pronounced differences between the two samples of compound C are observed in the region of the spin state marker of the spectrum (cm-1).
A comparison of the spectra in Figure 4 shows that the resonance Raman spectrum of the two samples of Compound C (Fig. 4b,c) is almost identical to that. Before attempting the low temperature experiments, we examined a sample of the 428/580 nm species at 277 K. The frequency of the intense oxidation state marker band at 1374 cm-1 is consistent with a Fea.
The resulting RR spectrum is almost identical to the spectrum of the resting enzyme shown in Figure 3a.
