Oxido-reductive titrations of cytochrome c oxidase followed by EPR spectroscopy

Experiments are described on oxido-reductive titrations of cytochrome c oxidase as followed by low-temperature EPR and reflectance spectroscopy. The reductants were cytochrome c or NADH and the oxidant ferricyanide. Experiments were conducted in the presence and absence of either cytochrome c or carbon monoxide, or both. An attempt is made to provide a complete quantitative balance of the changes observed in the major EPR signals. During reduction, the maximal quantity of heme represented in the high-spin ferric heme signals (g ～ 6; 2) is 25% of the total heme present, and during reoxidation 30%. With NADH reduction there is little difference between the pattern of disappearance of the low-spin ferric heme signals in the absence or presence of cytochrome c. The copper and high-spin heme signals, however, disappear at higher titrant concentrations in the presence of cytochrome c than in its absence. In these titrations, as well as in those with ferrocytochrome c, the quantitative balance indicates that, in addition to EPR-detectable components, EPR-undetectable components are also reduced, increasingly so at higher titrant concentrations. The quantity of EPR-undetectable components reduced appears to be inversely related to pH. A similar inverse relationship exists between pH and appearance of high-spin signals during the titration. At pH 9.3 the quantity of heme represented in the high-spin signals is < 5%, whereas it approximately doubles from pH 7.4 to pH 6.1. In the presence of CO less of the low-spin heme and copper signals disappears for the same quantity of titrant consumed, again implying reduction of EPR undetectable components. At least one of these components is represented in a broad absorption band centered at 655 nm. The stoichiometry observed on reoxidation, particularly in the presence of CO, is not compatible with the notion that the copper signal represents 100% of the active copper of the enzyme as a pair of interacting copper atoms.     

Kinetic studies on cytochrome c oxidase by combined epr and reflectance spectroscopy after rapid freezing.

1. Techniques and experiments are described concerned with the millisecond kinetics of EPT-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O2 or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy. 2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (less than 50 ms) approx. 0.5 electron equivalent per heme a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (less than 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a. 3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O2 in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O2 side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates. 4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms., whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO. 5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C.R., Hansen, R.E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477-2481). Both the low-spin (g=o; 2.2; 1.5) and slowly appearing high-spin (g=6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a3, when it is present in a state of interaction with EPR-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.     

Kinetic studies on cytochrome c oxidase by combined EPR and reflectance spectroscopy after rapid freezing

1. Techniques and experiments are described concerned with the millisecond kinetics of EPR-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O₂ or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy.2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (< 50 ms) approx. 0.5 electron equivalent per hame a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (> 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a.3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O₂ in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O₂ side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates.4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms, whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO.5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C. R., Hansen, R. E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477–2481). Both the low-spin (g = 3; 2.2; 1.5) and slowly appearing high-spin (g = 6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a₃, when it is present in a state of interaction with EPR-undetectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.     

The influence of pH on the EPR and redox properties of cytochrome c oxidase in detergent solution and in phospholipid vesicles

The EPR signals of oxidized and partially reduced cytochrome oxidase have been studied at pH 6.4, 7.4, and 8.4. Isolated cytochrome oxidase in both non-ionic detergent solution and in phospholipid vesicles has been used in reductive titrations with ferrocytochrome c.The g values of the low- and high-field parts of the low-spin heme signal in oxidized cytochrome oxidase are shown to be pH dependent. In reductive titrations, low-spin heme signals at g 2.6 as well as rhombic and nearly axial high-spin heme signals are found at pH 8.4, while the only heme signals appearing at pH 6.4 are two nearly axial g 6 signals. This pH dependence is shifted in the vesicles.The g 2.6 signals formed in titrations with ferrocytochrome c at pH 8.4 correspond maximally to 0.25–0.35 heme per functional unit (aa₃) of cytochrome oxidase in detergent solution and to 0.22 heme in vesicle oxidase. The total amount of high-spin heme signals at g 6 found in partially reduced enzyme is 0.45–0.6 at pH 6.4 and 0.1–0.2 at pH 8.4. In titrations of cytochrome oxidase in detergent solution the g 1.45 and g 2 signals disappear with fewer equivalents of ferrocytochrome c added at pH 8.4 compared to pH 6.4.The results indicate that the environment of the hemes varies with the pH. One change is interpreted as cytochrome a₃ being converted from a high-spin to a low-spin form when the pH is increased. Possibly this transition is related to a change of a liganded H₂O to OH^? with a concomitant decrease of the redox potential. Oxidase in phosphatidylcholine vesicles is found to behave as if it experiences a pH, one unit lower than that of the medium.     

Studies on the bacterial hemoglobin fromVitreoscilla

Summary Vitreoscilla contained a homodimeric bacterial hemoglobin (VtHb). The purification of this protein yielded VtmetHb which exhibited electronic and electron paramagnetic resonance (EPR) spectra, showing that it existed predominantly in a high-spin ferric form, both axial and rhombic components being present. The preparations also contained variable amounts of low-spin components. There was no evidence that these high-spin and low-spin forms were in equilibrium. The former were reducible by NADH catalyzed by the NADH-metVtHb reductase, and the latter were not. High ionic strength and high pH led to the formation of low-spin metVtHb; both treatments were reversible. Cyanide and imidazole liganded to VtHb resulted in the conversion of high-spin to low-spin ferric heme centers, each with characteristic electronic and EPR spectra. Some preparations of VtHb exhibited EPR signals consistent with a sulfur ligand bound to the ferric site. When VtHb was treated with NADH plus the reductase in the presence of oxygen, the intensity of the high-spin EPR signals decreased significantly. No reduction occurred in the absence of oxygen, suggesting a possible role for the superoxide anion. Dithionite treatment of VtHb resulted in a slow reduction, but the main product of the reaction of dithionite-reduced VtHb with oxygen was VtmetHb, not VtHbO₂. EPR spectra of whole cells ofVitreoscilla exhibited a variety of intense signals at low and high magnetic field, theg-values being consistent with the presence of high-spin ferric heme proteins, in addition to an iron-containing superoxide dismutase (FeSOD) and iron-sulfur proteins. EPR spectra of the cytosol fraction ofVitreoscilla showed the expected resonances for VtmetHb and FeSOD.Abbreviations A absorbance - DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetate - EPR electron paramagnetic resonance - HiPIP high-potential iron protein - SDS sodium dodecyl sulfate - SOD superoxide dismutase - VtHb Vitreoscilla hemoglobin - VtmetHb oxidizedVitreoscilla hemoglobin - VtHbO₂ oxygenatedVitreoscilla hemoglobin     

Oxidation-reduction potentials of the hemes in cytochrome C3 from Desulfovibrio gigas in the presence and absence of ferredoxin by EPR spectroscopy.

1. Ferricytochrome c3 from D. gigas exhibits two low-spin ferric heme EPR resonances with gz-values at 2.959 and 2.853. Ferrocytochrome c3 is diamagnetic based on the absence of any EPR signals. 2. EPR potentiometric titrations result in the resolution of the two low-spin ferric heme resonances into two additional heme components representing in total the four hemes of the cytochrome, with EM values of -235 mV and -315 mV at heme resonance I and EM values of -235 mV and -306 mV at heme resonance II. 3. EPR spectroscopy has detected a significant diminution of intensity (approx. 60 p. 100) in the gx amplitude of ferricytochrome c3 in the presence of D. gigas ferredoxin II. The presence of ferredoxin II also causes a more negative shift in the EM of the second components of the signals at heme resonances I and II of cytochrome C3. Both observations suggest that an interaction has occurred between cytochrome C3 and ferredoxin II. 4. The results presented suggest that the heme ligand environment of ferricytochrome c3 from D. gigas is less perturbed and/or less asymmetric than environment for ferricytochrome c3 from D. vulgaris whose EPR behavior indicates the non-equivalence of all four hemes.     

The electronic state of heme in cytochrome oxidase II. Oxidation-reduction potential interactions and heme iron spin state behavior observed in reductive titrations.

Magnetic circular dichroism (MCD), electron paramagnetic resonance (EPR), and optical absorption spectroscopies have been used to monitor the concentrations of oxidized and reduced heme and copper during stoichiometric reductive titrations of purified beef heart cytochrome oxidase. The MCD data are deconvoluted to obtain the concentrations of reduced cytochromes a and a3 during the titrations; analysis of the EPR spectra provides complementary data on the concentrations of the EPR-detectable species. For the native enzyme in the absence of exogenous ligands, cytochromes a and a3 are reduced to approximately the same extent at all points in the titration. The reduction of the EPR-detectable copper, on the other hand, initially lags the reduction of the two cytochromes but in the final stages of the titration is completely reduced prior to either cytochrome a or a3. These non-Nernstian titration results are interpreted to indicate that the primary mode of heme-heme interaction in cytochrome oxidase involves shifts in oxidation-reduction potential for each of the two cytochromes such that a change in oxidation state for one of the hemes lowers the oxidation-reduction potential of the second heme by approximately 135 mV. In these titrations high spin species are detected which account for 0.25 spin/oxidase maximally. Evidence is presented to indicate that at least some of these signals can be attributed to cytochrome a3+ which has undergone a low-spin to high-spin state transition in the course of the titration. In the presence of carbon monoxide the oxidation-reduction properties of cytochromes a and a3 are markedly altered. The a32+. CO complex is fully formed prior to reduction of either cytochrome a3+ or the EPR-detectable copper. The g = 3 EPR signal attributed to cytochrome a3+ decreases as the MCD intensity of cytochrome a2+ increases; no significant high-spin intensity is observed at any intermediate stage of reduction. We interpret these Nernstian titration results to indicate that in the presence of ligands the oxidation-reduction potential of cytochrome a relative to cytochrome a3 is determined by the oxidation-reduction state of the stabilized cytochrome a3 ligand complex; if ligand binding occurs to reduced cytochrome a3 then cytochrome a titrates with a lower potential; cytochrome a titrates with a higher potential if oxidized cytochrome a3 is stabilized by ligand binding.     

A cytochrome aa3-type quinol oxidase from Desulfurolobus ambivalens, the most acidophilic archaeon

Abstract Membranes of the extremely thermoacidophilic archaeon Desulfurolobus ambivalens grown under aerobic conditions contain a quinol oxidase of the cytochrome aa ₃-type as the most prominent hemoprotein. The partially purified enzyme consists of three polypeptide subunits with apparent molecular masses of 40, 27 and 20 kDa and contains two heme A molecules and one copper atom. CO difference spectra suggest one heme to be a heme a ₃-centre. The EPR spectra indicate the presence of a low-spin and a high-spin heme species. Redox titrations of the solubilized enzyme show the presence of two reduction processes, with apparent potentials of + 235 and + 330 mV. The enzyme cannot oxidize reduced cytochrome c , but rather serves as an oxidase of caldariella quinone. Due to their very simple composition, D . ambivalens cell appear as a promising candidate to study Structure-function relationships of cytochrome aa ₃ in the integral membrane state.     

The binding of carbon monoxide to cytochrome c oxidase.

The effect of CO on the optical absorbance spectrum of partially reduced cytochrome c oxidase has been studied. The changes at 432 and 590 nm suggest that the cytochrome alpha2/3+ - CO compound is formed preferentially and that concomitantly a second electron is taken up by the enzyme. From the CO-induced changes at 830 nm it is concluded that in the partially reduced enzyme addition of CO causes reoxidation of the copper component of cytochrome c oxidase. Addition of CO to partially reduced enzyme (2 electrons per 4 metal ions) also brings about a decrease in the intensities of electron paramagnetic resonance signals of high-spin heme iron near g = 6 and of the low-spin heme at g = 2.6. Concomitantly both the low-spin heme a signal at g = 3 and the copper signal at g = 2 increase in intensity. These results demonstrate that formation of the reduced diamagnetic cytochrome a3 - CO compound is accompanied by reoxidation of both the copper component detectable by electron paramagnetic resonance and possibly also by cytochrome a.     

EPR studies of cytochrome aa3 from Sulfolobus acidocaldarius. Evidence for a binuclear center in archaebacterial terminal oxidase.

The purified cytochrome aa3-type oxidase from Sulfolobus acidocaldarius (DSM 639) consists of a single subunit, containing one low-spin and one high-spin A-type hemes and copper [Anemüller, S. and Sch?fer, G. (1990) Eur. J. Biochem. 191, 297-305]. The enzyme metal centers were investigated by electron paramagnetic resonance spectroscopy (EPR), coupled to redox potentiometry. The low-spin heme EPR signal has the following g-values: gz = 3.02, gy = 2.23 and gx = 1.45 and the high-spin heme exhibits an almost axial spectrum (gy = 6.03 and gx = 5.97, E/D < 0.002). In the enzyme as isolated the low-spin resonance corresponds to 95 +/- 10% of the enzyme concentration, while the high-spin signal accounts for only 40 +/- 5%. However, taking into account the redox potential dependence of the high-spin heme signal, this value also rises to 95 +/- 10%. The high-spin heme signal of the Sulfolobus enzyme shows spectral characteristics distinct from those of the Paracoccus denitrificans one: it shows a smaller rhombicity (gy = 6.1 and gx = 5.9, E/D = 0.004 for the P. denitrificans enzyme) and it is easier to saturate, having a half saturation power of 148 mW compared to 360 mW for the P. denitrificans protein, both at 10 K. The EPR spectrum of an extensively dialyzed and active enzyme sample containing only one copper atom/enzyme molecule does not display CuA-like resonances, indicating that this enzyme contains only a CUB-type center. The EPR-redox titration of the high-spin heme signal, which is assigned to cytochrome a3, gives a bell shaped curve, which was simulated by a non-interactive two step redox process, with reduction potentials of 200 +/- 10 mV and 370 +/- 10 mV at pH = 7.4. The decrease of the signal amplitude at high redox potentials is proposed to be due to oxidation of a CUB(I) center, which in the CUB(II) state is tightly spin-coupled to the heme a3 center. The reduction potential of the low-spin resonance was determined using the same model as 305 +/- 10 mV at pH = 7.4 by EPR redox titration. Addition of azide to the enzyme affects only the high-spin heme signal, consistent with the assignment of this resonance to heme a3. The results are discussed in the context of the redox center composition of quinol and cytochrome c oxidases.     

Membrane tetraheme cytochrome c(m552) of the ammonia-oxidizing nitrosomonas europaea: a ubiquinone reductase

Cytochrome c(m552) (cyt c(m552)) from the ammonia-oxidizing Nitrosomonas europaea is encoded by the cycB gene, which is preceded in a gene cluster by three genes encoding proteins involved in the oxidation of hydroxylamine: hao, hydroxylamine oxidoreductase; orf2, a putative membrane protein; cycA, cyt c(554). By amino acid sequence alignment of the core tetraheme domain, cyt c(m552) belongs to the NapC/TorC family of tetra- or pentaheme cytochrome c species involved in electron transport from membrane quinols to a variety of periplasmic electron shuttles leading to terminal reductases. However, cyt c(m552) is thought to reduce quinone with electrons originating from HAO. In this work, the tetrahemic 27 kDa cyt c(m552) from N. europaea was purified after extraction from membranes using Triton X-100 with subsequent exchange into n-dodecyl beta-d-maltoside. The cytochrome had a propensity to form strong SDS-resistant dimers likely mediated by a conserved GXXXG motif present in the putative transmembrane segment. Optical spectra of the ferric protein contained a broad ligand-metal charge transfer band at approximately 625 nm indicative of a high-spin heme. Mossbauer spectroscopy of the reduced (57)Fe-enriched protein revealed the presence of high-spin and low-spin hemes in a 1:3 ratio. Multimode EPR spectroscopy of the native state showed signals from an electronically interacting high-spin/low-spin pair of hemes. Upon partial reduction, a typical high-spin heme EPR signal was observed. No EPR signals were observed from the other two low-spin hemes, indicating an electronic interaction between these hemes as well. UV-vis absorption data indicate that CO (ferrous enzyme) or CN(-) (ferric or ferrous enzyme) bound to more than one and possibly all hemes. Other anionic ligands did not bind. The four ferrous hemes of the cytochrome were rapidly oxidized in the presence of oxygen. Comparative modeling, based on the crystal structure and conserved residues of the homologous NrfH protein from Desulfovibrio of cyt c(m552), predicted some structural elements, including a Met-ligated high-spin heme in a quinone-binding pocket, and likely axial ligands to all four hemes.     

EPR signals from cytochrome c oxidase.

1. The major EPR signals from native and cytochrome c-reduced beef heart cytochrome c oxidase (EC 1.9.3.1) are characterized with respect to resonance parameters, number of components and total integrated intensity. A mistake in all earlier integrations and simulations of very anisotropic EPR signals is pointed out. 2. The so-called Cu2+ signal is found to contain at least three components, one "inactive" form and two nearly similar active forms. One of the latter forms, corresponding to about 20% of the total EPR detectable Cu, has not been observed earlier and can only be resolved in 35 GHz spectra. It is not reduced by cytochrome c and is thought to reflect some kind of inhomogeneity in the enzyme preparation. The 35 GHz spectrum of the cytochrome c reducible component shows a rhombic splitting and can be well simulated with g-values 2.18, 2.03 and 1.99. The origin of such a unique type of Cu2+ spectrum is discussed. 3. The low-spin heme signal in the oxidized enzyme (g = 3.03, 2.21, 1.45) is found to correspond closely to one heme and shows no signs of interaction with other paramagnetic centres. 4. The high-spin heme signals appearing in partly reduced oxidase are found to consist of at least three species, one axial and two rhombic types. An integration procedure is described that allows the determination of the total integral intensity of high-spin heme EPR signals only by considering the g = 6 part of the signals. In a titration with ascorbate and cytochrome c the maximum intensity of the g = 6 species corresponds to 23% of the enzyme concentration.     

Electron paramagnetic resonance studies of Pseudomonas cytochrome c peroxidase

The EPR spectrum at 15 K of Pseudomonas cytochrome c peroxidase, which contains two hemes per molecule, is in the totally ferric form characteristic of low-spin heme giving two sets of g-values with gz 3.26 and 2.94. These values indicate an imidazole-nitrogen : heme-iron : methionine-sulfur and an imidazole-nitrogen : heme-iron : imidazole-nitrogen hemochrome structure, respectively. The spectrum is essentially identical at pH 6.0 and 4.6 and shows only a very small amount of high-spin heme iron (g 5--6) also at 77 K. Interaction between the two hemes is shown to exist by experiments in which one heme is reduced. This induces a change of the EPR signal of the other (to gz 2.83, gy 2.35 and gx 1.54), indicative of the removal of a histidine proton from that heme, which is axially coordinated to two histidine residues. If hydrogen peroxide is added to the partially reduced protein, its EPR signal is replaced by still other signals (gz 3.5 and 3.15). Only a very small free radical peak could be observed consistent with earlier mechanistic proposals. Contrary to the EPR spectra recorded at low temperature, the optical absorption spectra of both totally oxidized and partially reduced enzyme reveal the presence of high-spin heme at room temperature. It seems that a transition of one of the heme c moieties from an essentially high-spin to a low-spin form takes place on cooling the enzyme from 298 to 15 K.     

The active form of the ferric heme in neutrophil cytochrome b(558) is low-spin in the reconstituted cell-free system in the presence of amphophil.

The spin state of the heme in superoxide (O(2)(.)(-))-producing cytochrome b(558) purified from pig neutrophils was examined by means of room-temperature magnetic circular dichroism (MCD) under physiological conditions. Cytochrome b(558) with varying amounts of low-spin and high-spin heme was prepared by either pH adjustment or heat treatment, and the O(2)(.)(-)-forming activity in a cell-free system was found to correlate with the low-spin heme content. The possibility that the O(2)(.)(-)-forming activity results from a transient high-spin ferric heme form that is induced during activation by anionic amphophils has also been investigated. EPR spectra of cytochrome b(558) activated by either arachidonic acid or myristic acid, showed that a transient high-spin ferric species accounting for approximately 50% of the heme appeared in the presence of arachidonic acid, but not in the presence of myristic acid. Hence the appearance of a transient high-spin ferric heme species on activation with an amphophil does not afford a common activation mechanism in the NADPH oxidase system. The EPR results for cytochrome b(558) activated with arachidonic acid showed that the transient high-spin ferric heme can bind cyanide. However, the high-spin ferric heme does not contribute to the O(2)(.)(-) production of cytochrome b(558) in cell-free assays in the presence of cyanide.     

Active site structure of SoxB-type cytochrome bo3 oxidase from thermophilic Bacillus

Two-subunit SoxB-type cytochrome c oxidase in Bacillus stearothermophilus was over-produced, purified, and examined for its active site structures by electron paramagnetic resonance (EPR) and resonance Raman (RR) spectroscopies. This is cytochrome bo3 oxidase containing heme B at the low-spin heme site and heme O at the high-spin heme site of the binuclear center. EPR spectra of the enzyme in the oxidized form indicated that structures of the high-spin heme O and the low-spin heme B were similar to those of SoxM-type oxidases based on the signals at g=6.1, and g=3.04. However, the EPR signals from the CuA center and the integer spin system at the binuclear center showed slight differences. RR spectra of the oxidized form showed that heme O was in a 6-coordinated high-spin (nu3 = 1472 cm(-1)), and heme B was in a 6-coordinated low-spin (nu3 = 1500 cm(-1)) state. The Fe2+-His stretching mode was observed at 211 cm(-1), indicating that the Fe2+-His bond strength is not so much different from those of SoxM-type oxidases. On the contrary, both the Fe2+-CO stretching and Fe2+-C-O bending modes differed distinctly from those of SoxM-type enzymes, suggesting some differences in the coordination geometry and the protein structure in the proximity of bound CO in cytochrome bo3 from those of SoxM-type enzymes.     

Biochemical and biophysical studies on cytochrome c oxidase. XVII. An epr study of the photodissociation of cytochrome a32+ · CO

1. The photodissociation reaction of the cytochrome c oxidase-CO compound was studied by EPR at 15 °K. Illumination with white light at both room and liquid N₂ temperatures of the partially reduced cytochrome c oxidase (2 electrons per 4 metals) in the presence of CO, causes the appearance of a rhombic (g_x = 6.60, g_y = 5.37) high-spin heme signal.This signal disappears completely upon darkening of the sample and reappears upon illumination at room temperature; accordingly the photolytic process is reversible. Under these conditions, no great changes in the intensities are observed, neither of the copper signal at g = 2, nor of the low-spin heme signal at g = 3, 2.2 and 1.5.2. In the presence of ferricyanide (2 mM) and CO, both the low-spin heme signal (g = 3.0, 2.2 and 1.5) and the copper signal of the partially reduced enzyme have intensities about equal to those of the completely oxidized enzyme in the absence of CO. Upon illumination of the carboxy-cytochrome c oxidase in the presence of ferricyanide, it was found that the rhombic high-spin heme signal appears without affecting appreciably the copper of low-spin heme signals. Thus, in the presence of ferricyanide the EPR-detectable paramagnetism of the illuminated carboxy-cytochrome c oxidase is higher than in the untreated oxidized enzyme.3. The membrane-bound cytochrome c oxidase reduced with NADH in the presence of CO and subsequently oxidized with ferricyanide shows a similar rhombic high-spin heme signal (g_x = 6.62, g_y = 5.29) upon illumination at room temperature. This signal disappears completely upon darkening and reappears upon illumination at room temperature.     

Hexaheme nitrite reductase from Desulfovibrio desulfuricans. M?ssbauer and EPR characterization of the heme groups

M?ssbauer and EPR spectroscopy were used to characterize the heme prosthetic groups of the nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774), which is a membrane-bound multiheme cytochrome capable of catalyzing the 6-electron reduction of nitrite to ammonia. At pH 7.6, the as-isolated enzyme exhibited a complex EPR spectrum consisting of a low-spin ferric heme signal at g = 2.96, 2.28, and 1.50 plus several broad resonances indicative of spin-spin interactions among the heme groups. EPR redox titration studies revealed yet another low-spin ferric heme signal at g = 3.2 and 2.14 (the third g value was undetected) and the presence of a high-spin ferric heme. M?ssbauer measurements demonstrated further that this enzyme contained six distinct heme groups: one high-spin (S = 5/2) and five low-spin (S = 1/2) ferric hemes. Characteristic hyperfine parameters for all six hemes were obtained through a detailed analysis of the M?ssbauer spectra. D. desulfuricans nitrite reductase can be reduced by chemical reductants, such as dithionite or reduced methyl viologen, or by hydrogenase under hydrogen atmosphere. Addition of nitrite to the fully reduced enzyme reoxidized all five low-spin hemes to their ferric states. The high-spin heme, however, was found to complex NO, suggesting that the high-spin heme could be the substrate binding site and that NO could be an intermediate present in an enzyme-bound form.     

A change in the heme stereochemistry of cytochrome c upon addition of sodium dodecyl sulfate: electron paramagnetic resonance and electronic absorption spectral study

Electron paramagnetic resonance and electronic absorption spectral changes upon addition of sodium dodecyl sulfate (SDS) to ferric and ferrous cytochrome c have been measured at 77 degrees K and at room temperature. The spectral changes upon addition of SDS to ferric cytochrome c were performed, in two steps, from native low-spin to another low-spin spectrum and subsequently to high-spin-like spectrum. On the other hand, the spectral changes upon addition of SDS to ferrous cytochrome c proceeded, in one step, from native low-spin to high-spin spectrum. The high-spin-like spectrum of ferric cytochrome c and the high-spin spectrum of ferrous cytochrome c in the presence of high concentrations of SDS are, respectively, apparently similar to those of ferric and ferrous cytochrome c' at physiological pH in spectral features. These spectral similarities suggest the similarities in the heme stereochemistry and the ground state of heme iron. Further, the spectra of cytochrome c in the presence of SDS varied with the change of pH values. The ferric high-spin-like and ferrous high-spin spectra were stable at neutral pH and below it. Conformational changes of cytochrome c upon addition of SDS are also discussed.     

Protein conformational perturbations affect the photoreduction of native cytochrome c peroxidase (III) at alkaline pH.

Ferric cytochrome c peroxidase (CCP) undergoes a ligation-state transition from a pentacoordinate, high-spin (5c/hs) heme to a hexacoordinate, low-spin (6c/1s) heme when titrated over a pH range of 7.30-9.70. This behavior is similar to that exhibited by the ferrous form of the enzyme. However, the photodissociation of the low-spin, axial ligand, exhibited by ferrous CCP at alkaline pH, is not observed for ferric CCP. Instead, a photoinduced reduction of the ferric heme is apparent in the pH range 7.90-9.70. In the absence of O2 and redox mediators such as methyl viologen (MV2+), the reoxidation of the photoreduced enzyme is very slow (tau 1/2 approximately 3 min). F(-)-bound CCP(III) (6c/hs) displays similar pH-dependent photoreduction. Horseradish peroxidase, however, does not. The formation of 6c/1s heme coincides with the onset of appreciable photoreduction (between laser pulses, > 60 ms) of CCP (III) at alkaline pH, suggesting a global protein conformational rearrangement within or around its heme pocket. Photoreduction of alkaline CCP(III) most likely involves intramolecular electron transfer (ET) from the aromatic residue in the proximal heme pocket to the photoexcited heme. We speculate that the kinetics of electron transfer are affected by changes in the orientation of Trp-191.     

The effect of phospholipid depletion on the EPR behavior of cytochrome oxidase

Phospholipids are essential components for electron transport activity of cytochrome oxidase. Recently, we have found that the removal of phospholipids from the oxidase affected the copper and low-spin heme signals, and conceivably other paramagnetic centers as demonstrated by EPR spectroscopy. At 4.2–30 °K, the signal amplitudes and power saturation behaviors were studied at approximately g = 2.0 for the copper signal, and in the neighborhood of g = 3.0 for the low-spin heme signal. After depletion of phospholipids the amplitude of the copper signal decreased 25–30% at 12–30 °K and below 12 °K 40–50% under nonsaturating conditions. The amplitude of the low-spin heme signal decreased 60–70% at 4.2–20 °K. Below 14 °K both signals became more resistant to power saturation, but the copper signal was more readily saturated above this temperature, compared to the oxidase with about 25% lipid. After removal of phospholipids, the spectral features of the copper signal remained essentially the same, but the low-spin heme signal broadened and became very asymmetric to show two signals as revealed by the second harmonic EPR spectra. These findings may explain, at least partially, the wide variations in percentage of EPR detectable copper and heme of cytochrome oxidase reported by different laboratories. Unequivocally, the EPR behavior of cytochrome oxidase is not only affected by the protein moiety, but also by the associated phospholipids of the enzyme.