Effects of a thiolate axial ligand on the pi-->pi* electronic states of oxoferryl porphyrins: a study of the optical and resonance Raman spectra of compounds I and II of chloroperoxidase.

Optical absorption and resonance Raman spectra have been investigated for enzymatic intermediates, compounds I and II, of chloroperoxidase (CPO) which contains a thiolate-ligated iron porphyrin. Compound I of CPO (CPO-I), an oxoferryl porphyrin pi cation radical, gave an apparently asymmetric single-peaked Soret band at 367 nm, for which band fitting analyses revealed the presence of two transition bands around 365 and 415 nm. Compound II of CPO (CPO-II), an oxoferryl neutral porphyrin, gave a split Soret spectrum with two bands (blue and red Soret bands) at 373 and 436 nm. Thus both CPO-I and CPO-II can be categorized as hyperporphyrins. The maximum extinction coefficients (epsilon(b) and epsilon(r)) and energies (Eb and Er) of the blue and red Soret bands of CPO-II were found to fall on an epsilon(b)/epsilon(r) versus Eb-Er correlation line derived from data reported for six-coordinate ferrous derivatives of cytochrome P450 and CPO. Corresponding data for CPO-I did not fall on the correlation line. Resonance enhancement of the FeIV=O stretching (vFeO) Raman band was found for CPO-I when Raman scattering was excited at wavelengths within both transition bands around 365 and 415 nm, while the vFeO Raman band was not identified for CPO-II at any of the excitation wavelengths examined here. These findings suggest that the thiolate axial ligand causes Soret band splitting of CPO-II through configuration interaction between the sulfur-->porphyrin e(g)* charge transfer and porphyrin a1u,a2u-->e(g)* transitions, while the FeO portion is important in determining the shape of the Soret band of CPO-I.

Oxygen activation and reduction in respiration: involvement of redox-active tyrosine 244.

Cytochrome oxidase activates and reduces O(2) to water to sustain respiration and uses the energy released to drive proton translocation and adenosine 5'-triphosphate synthesis. A key intermediate in this process, P, lies at the junction of the O(2)-reducing and proton-pumping functions. We used radioactive iodide labeling followed by peptide mapping to gain insight into the structure of P. We show that the cross-linked histidine 240-tyrosine 244 (His240-Tyr244) species is redox active in P formation, which establishes its structure as Fe(IV) = O/Cu(B)2+-H240-Y244. Thus, energy transfer from O2 to the protein moiety is used as a strategy to avoid toxic intermediates and to control energy utilization in subsequent proton-pumping events.

From water to oxygen and back again: mechanistic similarities in the enzymatic redox conversions between water and dioxygen.

We propose that the interconversions of water and oxygen are catalyzed by the transition metal ions of Photosystem II and cytochrome c oxidase in remarkably similar ways. Oxygen-oxygen bond formation and cleavage occurs between two oxygen atoms that are bound as terminal ligands to two redox-active metal ions. Hydrogen atom transfer to or from a tyrosine residue is an essential component of the processes in both enzymes.

Dioxygen activation and bond cleavage by mixed-valence cytochrome c oxidase.

Elucidating the structures of intermediates in the reduction of O2 to water by cytochrome c oxidase is crucial to understanding both oxygen activation and proton pumping by the enzyme. In the work here, the reaction of O2 with the mixed-valence enzyme, in which only heme a3 and CuB in the binuclear center are reduced, has been followed by time-resolved resonance Raman spectroscopy. The results show that O==O bond cleavage occurs within the first 200 micros after reaction initiation; the presence of a uniquely stable Fe---O---O(H) peroxy species is not detected. The product of this rapid reaction is a heme a3 oxoferryl (FeIV==O) species, which requires that an electron donor in addition to heme a3 and CuB must be involved. The available evidence suggests that the additional donor is an amino acid side chain. Recent crystallographic data [Yoshikawa, S., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., Yamashita, E., Inoue, N., Yao, M., Fei, M. J., Libeu, C. P., Mizushima, T., et al. Science, in press; Ostermeier, C., Harrenga, A. , Ermler, U. & Michel, H. (1997) Proc. Natl. Acad. Sci. USA 94, 10547-10553] show that one of the CuB ligands, His240, is cross-linked to Tyr244 and that this cross-linked tyrosyl is ideally positioned to participate in dioxygen activation. We propose a mechanism for O---O bond cleavage that proceeds by concerted hydrogen atom transfer from the cross-linked His---Tyr species to produce the product oxoferryl species, CuB2+---OH-, and the tyrosyl radical. This mechanism provides molecular structures for two key intermediates that drive the proton pump in oxidase; moreover, it has clear analogies to the proposed O---O bond forming chemistry that occurs during O2 evolution in photosynthesis.

Resonance Raman/absorption characterization of the oxo intermediates of cytochrome c oxidase generated in its reaction with hydrogen peroxide: pH and H2O2 concentration dependence.

Effects of pH and H2O2 concentration on the reaction of cytochrome c oxidase (CcO) with H2O2 were studied with the high-performance Raman/absorption simultaneous determination technique reported previously (Proshlyakov et al., 1996). This reaction generates two intermediates called 607- and 580-nm forms, and we found that they show the same oxygen-isotope-sensitive RR bands as those of the intermediates in O2 reduction by CcO. In transient absorption spectra obtained under single turnover conditions, the 607-nm form appeared as the primary intermediate and subsequently the 580-nm and resting forms, suggesting that H2O2 serves as an oxidant for the resting enzyme but as a reductant for both the 607- and 580-nm forms in the peroxide cycle. The rise rate of absorption at 607 nm was insensitive to the H2O/D2O exchange, but the decay was significantly slower in D2O than in H2O. With the microcirculating system, each intermediate was maintained at a constant level under steady-state conditions by supplying H2O2 continuously. In the pH range between 7.4 and 10.0, the population of the 607-nm form decreased at higher pH and at higher concentrations of H2O2. The Fe=O stretching (VFe=O) frequencies of the oxo heme of the 607-nm form, observed at 804/769 cm-1 for their H2(16)O2/H2(18)O2 derivatives, were unaltered in this pH range and exhibited a D2O/H2O shift even at pH 10.0. This indicates that the iron-bound oxygen is hydrogen-bonded to a distal residue in this pH range. When the 580-nm form is dominant under the nonsaturating level of H2O2, two other oxygen-isotope-sensitive Raman bands have been observed at 785/750 cm-1 and 355/340 cm-1 at neutral pH, but the former disappeared above pH 8.5 and the latter above pH 9.0 without significant changes of absorption spectra, suggesting the presence of two separate species in the name of the 580-nm form. However, under the saturating concentration of H2O2, these Raman bands were unaltered between pH 7.4 and 10.0. In contrast, in the absence of excess peroxide, no oxygen-isotope-sensitive RR bands were observed despite dominance of the 580-nm form. The disappearance of these Raman bands demonstrates the occurrence of oxygen exchange between the oxo heme and bulk water, whose rate surpasses the formation rate of the 580-nm form at alkaline pH and/or at low H2O2 concentration. Such an oxygen exchange did not take place in the 607-nm form. Under the identical experimental conditions for generating a particular steady state, the exchange of H2O with D2O caused significant depopulation of the 580-nm form and concomitant increase of the 607-nm form. This was satisfactorily interpreted in terms of the difference in the decay rate of the 607-nm form between H2O and D2O. Thus, the reduction of the 607-nm form to the 580-nm form is likely to be a key step of the redox-linked proton pumping in the O2 reduction.

Microcirculating system for simultaneous determination of Raman and absorption spectra of enzymatic reaction intermediates and its application to the reaction of cytochrome c oxidase with hydrogen peroxide.

A new high-performance device for Raman/absorption simultaneous determination was developed. This was combined with a newly designed microcirculating system and was successfully applied to study intermediates in the reaction of bovine oxidized cytochrome c oxidase (CcO) with hydrogen peroxide under steady state conditions at ambient temperatures. Measurements with this device made it possible to correlate directly the species defined in terms of the visible absorption characteristics with specific Raman bands. The "607 nm" form of the enzyme obtained with H2(16)O2 gave an oxygen isotope sensitive band at 804 cm-1 (769 cm-1 with H2(18)O2) in the Soret excited resonance Raman (RR) spectrum. Its frequency and isotope frequency shifts are exactly the same as those observed previously with 607 nm excitation in nonsimultaneous measurements for the 607 nm form, for which the presence of an oxoiron heme was demonstrated. The so-called " 580 nm" form of the enzyme obtained with H2(16)O2 gave the main oxygen isotope sensitive band at 785 cm-1 (750 cm-1 with H2(18)O2) but appeared to consist of multiple species. This band was assigned to the FeIV = O stretching mode of ferryloxo heme on the basis of its isotopic frequency shift. Another oxygen isotope sensitive band was found at 355 cm-1 (340 cm-1 for H2(18)O2), similar to the case of dioxygen reaction. Temporal behavior of this band did not agree with either that of the 804 cm-1 band or that of the 785 cm-1 band but seemed to grow between the two species. The RR spectra in the higher frequency region of the 607 nm and 580 nm forms excited at 427 nm were quite alike and did not support the formation of a porphyrin pi-cation radical.

Selective resonance Raman observation of the "607 nm" form generated in the reaction of oxidized cytochrome c oxidase with hydrogen peroxide.

Resonance Raman spectra were measured selectively for the "607 nm" form, which had been assigned to a peroxy intermediate formed in the reaction of oxidized cytochrome c oxidase with hydrogen peroxide at ambient temperature. A single oxygen isotope-sensitive band was found at 803 cm-1 for the reaction with H2(16)O2 (at 769 cm-1 with H2(18)O2) upon excitation at 607 nm, the wavelength of the difference absorption maximum characteristic of the "peroxy" intermediate. Upon excitation at shorter wavelengths (down to 580 nm), the Raman spectrum simply became weaker without yielding any new features. When H2(16)O18O was used, two bands were observed at 803 and 769 cm-1 (within an accuracy of 0.5 cm-1), but with only half the intensity of those observed with H2(16)O2 or H2(18)O2, which ruled out the possibility that the 803 cm-1 band arose from the O-O or Fe-O2 stretching of the FeIII(O-O-) heme. Conversely, the 34-cm-1 downshift with 18O is in good agreement with the calculated 16O/18O shift (35 cm-1) expected for the diatomic Fe = 16O oscillator at 803 cm-1. This band exhibited an upshift by 1.3 cm-1 in 2H2O, similar to the case of compound II of horseradish peroxidase at neutral pH, and indicative of the presence of a hydrogen bond to the FeIV = O oxygen. The 803/769 cm-1 pair of resonance Raman bands were also observed upon blue excitation, as is the case for the bands found in the dioxygen cycle of this enzyme (Ogura, T., Takahashi, S., Hirota, S., Shinzawa-Itoh, K., Yoshikawa, S., Appelman, E. H., and Kitagawa, T. (1993) J. Am. Chem. Soc. 115, 8527-8536). This observation provides the first direct characterization of the 607 nm form of this enzyme in its reaction with H2O2.