Partial purification and characterization of two soluble c-type cytochromes from Chromatium vinosum

Two c-type cytochromes from Chromatium vinosum have been partially purified and characterized. Cytochrome c₅₅₀, which appears to function as an electron carrier in the cyclic electron transport chain of this photosynthetic purple sulfur bacterium, has a molecular weight of approximately 15,000 and an oxidation-reduction midpoint potential (E_m) of + 240 mV at pH 7.4. It has (in the reduced form) an α band at 550 nm and a β band at 520 nm. Cytochrome c₅₅₁ is characterized by absorbance maxima at 354 and 409 nm in the oxidized form and 418, 523, and 551 nm in the reduced form. The reduced cytochrome reacts with CO. Cytochrome c₅₅₁ is a monomeric protein with a molecular weight of 18,800 ± 700 and E_m = ?299 ± 5 mV (pH independent between pH 6.3 and 8.0). It appears to lack a methionine axial ligand as indicated by the absence of an absorbance band at 695 nm in the oxidized form.     

pH Variation of midpoint potential for three photosynthetic bacterial cytochromes c′. A link between physical and functional properties

Midpoint redox potential (E_M) versus pH curves are reported over the pH range 5 to 10 for the cytochromes c′ from three species of purple photosynthetic bacteria: Rhodospirillum rubrum, Rhodopseudomonas palustris and Chromatium vinosum. In each case, theoretical curves are fitted to the data and pK values for the reduced (pH 5–5.5) and oxidized (pH 8–8.5) forms of the protein are found to influence the midpoint redox potentials. The oxidized form pK values in each case are found to correlate with previously determined pK values for variation in physical and/or spectroscopic properties. This correlation of functional and physical observables is discussed in terms of a possible mechanism of control of midpoint redox potential through heme iron-ligand bonding as moderated by the protein conformation in response to solution conditions. The reduced form pK values are discussed in terms of a mechanism which would alter the polarity of the heme environment, thereby influencing redox potentials.     

Thermodynamic and EPR characterization of mitochondrial succinate-cytochrome c reductase-phospholipid complexes

Succinate-cytochrome c reductase can be easily solubilized in a phospholipid mixture (1:1, lysolecithin:lecithin) in the absence of detergents. The resulting solution contains two b cytochromes with half-reduction potentials of 95 ± 10 mV (b₅₆₁), and 0 ± 10 mV (b₅₆₆) and cytochrome c₁ (E_{m 7.2} = +280±5 mV). The oxidation-reduction midpoint potentials obtained by optical potentiometric titrations are identical to those determined by the EPR titrations and are 40–60 mV higher than the corresponding midpoint potentials of these cytochromes in intact mitochondria. In contrast to detergent-suspended preparations, no CO-sensitive cytochrome b can be detected in the phospholipid-solubilized preparation or intact mitochondria. The half-reduction potential of cytochrome b₅₆₆ is pH-dependent above pH 7.0 (?60 mV/pH unit) while that of b₅₆₁ is essentially pH-independent from pH 6.7–8.5, in contrast to its pH dependence in intact mitochondria. EPR characterizations show the presence of three oxidized low-spin heme-iron signals with g values of 3.78, 3.41 and 3.37. The identification of these signals with cytochromes b₅₆₆ (b_T), b₅₆₁ (b_K) and c₁ respectively is made on the basis of redox midpoint potentials. No significant amounts of oxidized high-spin heme-iron are detectable. In addition, the preparation contains four distinct types of iron-sulfur centers: S₁ and S₂ (E_{m 7.4} = ?260 mV and 0 mV), and two iron-sulfur proteins which are associated with the cytochrome b-c₁ complex: Rieske's iron-sulfur protein (E_{m 7.4} = +280 mV) and Ohnishi's Center 5 (E_{m 7.4} = +35 mV).     

Detection of two further b-type cytochromes in Rhodopseudomonas spheroides

The photosynthetically-incompetent mutant V-2 of Rhodopseudomonas spheroides which is incapable of synthesising bacteriochlorophyll was grown aerobically under conditions of both high and low aeration. Potentiometric titration at 560 nm minus 570 nm revealed the presence of several different components tentatively identified as b-type cytochromes. Two such components of oxidation-reduction midpoint potentials of +390 mV ± 10 mV and +255 mV ± 7 mV have not previously been detected in membranes of Rps. spheroides. These components have also been resolved by difference spectra at controlled oxidation-reduction potentials and fourth derivative spectra. Neither component appeared to react with CO. With increasing aeration of the culture medium the relative concentration of these two b-type cytochromes diminished, whilst that of the a-type oxidase increased.     

Effect of oxidation-reduction potential on light-induced cytochrome and bacteriochlorophyll reactions in chromatophores from the photosynthetic green bacterium Chlorobium

The reaction center bacteriochlorophyll of Chlorobium thiosulfatophilum has a midpoint oxidation-reduction potential (E_m) of +330 mV. Its photooxidation is unaffected by oxidation-reduction potentials in the range from +260 mV to ?70 mV but on further reduction is attenuated to zero in a one-electron transition with an E_m of ?130 mV.A c-type cytochrome with an E_m of +220 mV and absorption maxima at 551–552 nm (α-band) and 420 nm (γ-band) is present in Chlorobium chromatophores and undergoes photooxidation. Cytocrome c photooxidation is attenuated to zero in two 1-electron steps with E_m of +30 mV and ?130 mVPossible roles for +30 mV and ?130 mV components in photosynthetic electron transport in Chlorobium are discussed.     

Biochemical and biophysical studies on cytochrome c oxidase XVI. Circular dichroic study of cytochrome c oxidase and its ligand complexes

1. CD spectra of cytochrome c oxidase have been determined both in the absence and presence of the extrinsic ligands CO, NO, cyanide and azide.2. CO and NO affect the CD spectrum of cytochrome c oxidase in a similar way.3. Cyanide and azide also affect the CD spectrum of cytochrome c oxidase in a similar way, but distinctly different from CO and NO.4. From the CD spectra of the oxidized and reduced enzyme, in the presence and absence of extrinsic ligands, CD difference spectra (reduced minus oxidized) are calculated for the so-called cytochrome a and cytochrome a₃ moieties of the enzyme.5. These spectra are largely dependent on the extrinsic ligand used. It is therefore concluded that these spectra do not represent independent cytochrome a and cytochrome a₃ difference spectra, but that heme-heme interactions occur within the cytochrome c oxidase molecule, in such a way that binding of a ligand to one of the heme a groups of cytochrome c oxidase affects the spectral properties of the other heme a group.6. As a consequence, ligand-binding studies cannot give information as to the pre-existence of separate cytochrome a and cytochrome a₃ moieties in the absence of extrinsic ligands.     

The catalytic reaction of cytochrome c oxidase probed by in situ gas titrations and FTIR difference spectroscopy

Cytochrome c oxidase (CcO) is a transmembrane heme‑copper metalloenzyme that catalyzes the reduction of O₂ to H₂O at the reducing end of the respiratory electron transport chain. To understand this reaction, we followed the conversion of CcO from Rhodobacter sphaeroides between several active-ready and carbon monoxide-inhibited states via attenuated total reflection Fourier-transform infrared (ATR FTIR) difference spectroscopy. Utilizing a novel gas titration setup, we prepared the mixed-valence, CO-inhibited R₂CO state as well as the fully-reduced R₄ and R₄CO states and induced the “active ready” oxidized state O_H. These experiments are performed in the dark yielding FTIR difference spectra exclusively triggered by exposure to O₂, the natural substrate of CcO. Our data demonstrate that the presence of CO at heme a₃ does not impair the catalytic oxidation of CcO when the cycle starts from the fully-reduced states. Interestingly, when starting from the R₂CO state, the release of the CO ligand upon purging with inert gas yield a product that is indistinguishable from photolysis-induced states. The observed changes at heme a₃ in the catalytic binuclear center (BNC) result from the loss of CO and are unrelated to electronic excitation upon illumination. Based on our experiments, we re-evaluate the assignment of marker bands that appear in time-resolved photolysis and perfusion-induced experiments on CcO.     

Cytochrome b and photosynthetic sulfur bacteria

Chromatophores isolated from the purple sulfur bacterium Chromatium and the green sulfur bacterium Chlorobium exhibit absorbance changes in the cytochrome -band region consistent with the presence of a b-type cytochrome. Cytochrome content determined by reduced minus oxidized difference spectra and by heme analysis suggests that each bacterium contains one cytochrome b per molecule of photochemically active bacteriochlorophyll (reaction-center bacteriochlorophyll).

The b-type cytochrome in Chromatium has an -band maximum at 560 nm and a midpoint oxidation-reduction potential of −5 mV at pH 8.0. The b-type cytochrome in Chlorobium has an -band maximum at 564 nm and an apparent midpoint oxidation-reduction potential near −90 mV.

Chromatophores isolated from both Chromatium and Chlorobium cells catalyze a photoreduction of cytochrome b that is enhanced in the presence of antimycin A. Antimycin A and 2-n-heptyl-4-hydroxyquinoline-N-oxide inhibit endogenous (but not phenazine methosulfate-mediated) cyclic photophosphorylation in Chromatium chromatophores and non-cyclic electron flow from Na₂S to NADP in Chlorobium chromatophores. These observations suggest that b-type cytochromes may function in electron transport reactions in photosynthetic sulfur bacteria.     

Structural dynamics of proximal heme pocket in HemAT-Bs associated with oxygen dissociation

HemAT from Bacillus subtilis (HemAT-Bs) is a heme-containing O₂ sensor protein that acts as a chemotactic signal transducer. Binding of O₂ to the heme in the sensor domain of HemAT-Bs induces a conformational change in the protein matrix, and this is transmitted to a signaling domain. To characterize the specific mechanism of O₂-dependent conformational changes in HemAT-Bs, we investigated time-resolved resonance Raman spectra of the truncated sensor domain and the full-length HemAT-Bs upon O₂ and CO dissociation. A comparison between the O₂ and CO complexes provides insights on O₂/CO discrimination in HemAT-Bs. While no spectral changes upon CO dissociation were observed in our experimental time window between 10 ns and 100 μs, the band position of the stretching mode between the heme iron and the proximal histidine, ν(Fe–His), for the O₂-dissociated HemAT-Bs was lower than that for the deoxy form on time-resolved resonance Raman spectra. This spectral change specific to O₂ dissociation would be associated with the O₂/CO discrimination in HemAT-Bs. We also compared the results obtained for the truncated sensor domain and the full-length HemAT-Bs, which showed that the structural dynamics related to O₂ dissociation for the full-length HemAT-Bs are faster than those for the sensor domain HemAT-Bs. This indicates that the heme proximal structural dynamics upon O₂ dissociation are coupled with signal transduction in HemAT-Bs.     

Measurement of the oxidation reduction potential of the EPR detectable active centre of the molybdenum iron protein of Chromatium nitrogenase

The midpoint oxidation-reduction potential of the EPR detectable centre of the molybdenum iron protein of Chromatium nitrogenase has been measured. Two centres with identical EPR spectra but different midpoint potentials were detected. The measured midpoint potentials are (1) Em_7.5 = ?60 mV and (2) Em_7.5 = ?260 mV. The midpoint potentials were not affected by other components of the nitrogen fixing system.     

Heliobacterial Rieske/cytb complex

Data on structure and function of the Rieske/cytb complex from Heliobacteria are scarce. They indicate that the complex is related to the b ₆ f complex in agreement with the phylogenetic position of the organism. It is composed of a diheme cytochrome c, and a Rieske iron–sulfur protein, together with transmembrane cytochrome b ₆ and subunit IV. Additional small subunits may be part of the complex. The cofactor content comprises heme c _i, first discovered in the Q_i binding pocket of b ₆ f complexes. The redox midpoint potentials are more negative than in b ₆ f complex in agreement with the lower redox midpoint potentials (by about 150 mV) of its reaction partners, menaquinone, and cytochrome c ₅₅₃. The enzyme is implicated in cyclic electron transfer around the RCI. Functional studies are favored by the absence of antennae and the simple photosynthetic reaction chain but are hampered by the high oxygen sensitivity of the organism, its chlorophyll, and lipids.     

Optical measurements of intracellular oxygen concentration of rat heart in vitro

The optical characteristics of hemoglobin-free perfused rat heart have been examined in detail. Ethyl hydrogen peroxide is found to convert myoglobin into “ferryl compound” in the perfused heart, as is also seen in vitro. After pretreatment with ethyl hydrogen peroxide, a typical mitochondrial absorption spectrum, similar to that of isolated rat heart mitochondria, is obtained in perfused heart. The overall absorption spectrum of the heart obtained by the aerobic to anaerobic transition is a superposition of the mitochondrial spectrum on that of myoglobin. By comparing these spectra, it is found that measurement of cytochrome a + a₃ at 605–620 nm is possible in spite of the absorbance change due to the oxygenation-deoxygenation of myoglobin, whereas the wavelength pairs for cytochrome c at 550-540 nm, cytochrome b at 562–575 nm and cytochrome a + a₃ at 445–450 nm can not be used in the heart because of interference from the absorption change of myoglobin. The partial pressure of O₂ (P₅₀) which is required for half maximal deoxygenation (or oxygenation) of myoglobin in perfused heart is found to be 2.4 mm Hg at room temperature and the Hill constant, n, is 1.1; these values are similar to those of myoglobin purified from rat heart. The steady-state O₂ titration has been performed by using absorbancy changes of myoglobin and cytochrome a + a₃ as intracellular O₂ indicators. In the perfused heart, the percentage change of oxygenation-deoxygenation of myoglobin parallels the oxidation-reduction of cytochrome a + a₃, while the mixture of purified myoglobin and isolated mitochondria shows a deviation, reflecting the difference of O₂ affinities between myoglobin and cytochrome a + a₃. The results indicate that there may be an O₂ gradient between cytosolic and mitochondrial compartments in the hemoglobin-free perfused heart. The absorption changes of myoglobin and of cytochrome a + a₃ can be measured in a single contraction-relaxation cycle. A triple beam method was introduced to eliminate the effect of light scattering changes in these measurements. The results demonstrated that myoglobin is more oxygenated during the systolic and diastolic periods and deoxygenated in the resting period, whereas cytochrome a + a₃ is more reduced in systole and diastole and oxidized in the resting state. Changing the perfusion conditions greatly alters the time course of the events which occur during the contraction-relaxation cycle of the perfused heart.     

The pH dependence of the oxidation-reduction midpoint potential of cytochromes c2 in vivo

A recent report by Pettigrew et al. [Biochim. Biophys. Acta 430, (1976), 197–208] has examined the pH dependence of the oxidation-reduction midpoint potential of cytochromes c₂ in vitro. In media of low ionic strength, these workers identified several pKs on the oxidized forms of the cytochromes, and in some cases there were also pKs on the reduced species. In this work we examine the pH dependence of the midpoint potentials of the cytochromes in situ, attached to the chromatophore membrane. Under these conditions no pK values are detected, and we conclude that in vivo there is no net change in the protonation of cytochrome c₂ during oxidation or reduction.     

The effect of mitochondrial energization on cytochrome c oxidase kinetics as measured at low temperatures. I. The reaction with carbon monoxide

The kinetics of CO binding by the cytochrome c oxidase of pigeon heart mitochondria were studied as a function of membrane energization at temperatures from 180 to 280°K in an ethylene glycol/water medium. Samples energized by ATP showed acceleration of CO binding compared to those untreated or uncoupled by carbonylcyanide p-trifluoromethoxyphenylhydrazone but only at relatively low temperatures and high CO concentrations. Experiments using samples in a “mixed valency” (partially oxidized) state showed that the acceleration of ligand binding is not due to the formation of a partially oxidized state via reverse electron transport.It is concluded that in the deenergized state one CO molecule can be closely associated with the cytochrome a₃ heme site without actually being bound to the heme iron; in the energized state, two or more ligand molecules can occupy this intermediate position.The change in the apparent ligand capacity of a region near the heme iron in response to energization is evidence for an interaction between cytochrome oxidase and the ATPase system. Furthermore, these results suggest a control mechanism for O₂ binding.     

YhjA - An Escherichia coli trihemic enzyme with quinol peroxidase activity

The trihemic bacterial cytochrome c peroxidase from Escherichia coli, YhjA, is a membrane-anchored protein with a C-terminal domain homologous to the classical bacterial peroxidases and an additional N-terminal (NT) heme binding domain. Recombinant YhjA is a 50?kDa monomer in solution with three c-type hemes covalently bound. Here is reported the first biochemical and spectroscopic characterization of YhjA and of the NT domain demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P (active site), NT and E hemes were established to be ?170?mV, +133?mV and +210?mV, respectively, at pH?7.5. YhjA has quinol peroxidase activity in vitro with optimum activity at pH?7.0 and millimolar range K_M values using hydroquinone and menadiol (a menaquinol analogue) as electron donors (K_M?=?0.6?±?0.2 and 1.8?±?0.5?mM H₂O₂, respectively), with similar turnover numbers (k_cat?=?19?±?2 and 13?±?2?s^?1, respectively). YhjA does not require reductive activation for maximum activity, in opposition to classical bacterial peroxidases, as P heme is always high-spin 6-coordinated with a water-derived molecule as distal axial ligand but shares the need for the presence of calcium ions in the kinetic assays. Formation of a ferryl Fe(IV)?=?O species was observed upon incubation of fully oxidized YhjA with H₂O₂. The data reported improve our understanding of the biochemical properties and catalytic mechanism of YhjA, a three-heme peroxidase that uses the quinol pool to defend the cells against hydrogen peroxide during transient exposure to oxygenated environments.     

On the identity of the high spin heme components of cytochrome c oxidase

In oxidized, resting cytochrome c oxidase (EC 1.9.3.1) and under most conditions of partial reduction ? 50% of the heme components are detected by EPR spectroscopy. When the enzyme is fully reduced in the presence of equimolar quantities of cytochrome c, anaerobic reoxidation by an excess of a chemical oxidant (ferricyanide, porphyrexide) produces intense high and low spin heme signals simultaneously. The time range in which maximal high spin signals are observed is 0.1–2 s after mixing. Under these conditions 35–50% of the total heme a is accounted for by the low spin heme signal and 35–40% by the high spin signals, with the rhombic component accounting for 30–35% of the total heme. It is concluded that under these conditions, the major portion of both heme components must be EPR detectable. Thus, if the generally accepted assignment of the low spin signal to cytochrome a is adopted, it follows that in the experiments described, cytochrome a₃ is represented in the rhombic high spin signal. The quantities of heme represented in the axial high spin signal are too small for a definitive assignment; these signals could originate from either heme. When after formation of high spin signals as described, O₂ is admitted, the rhombic signal is eliminated within 4 ms. In the presence of the strongest rhombic high spin signals, the absorption band at 655 nm is only ? 25% developed. The implications of these findings are discussed in the context of present hypotheses concerning the state and interactions of cytochrome c oxidase components during oxidation-reduction.     

Polyethylene glycol promotes autoxidation of cytochrome c

Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of PEG. The redox potential of heme c was determined by the potentiometric titration to be +236?±?3?mV in the absence of PEG, which was negatively shifted to +200?±?4?mV in the presence of PEG. The underlying the rapid oxidation was explored by examining the structural changes in Cyt c in the presence of PEG using UV–visible absorption, circular dichroism, resonance Raman, and fluorescence spectroscopies. These spectroscopic analyses suggested that heme oxidation was induced by a modest tertiary structural change accompanied by a slight shift in the heme position (<1.0?Å) rather than by partial denaturation, as is observed in the presence of cardiolipin. The near-infrared spectra showed that PEG induced dehydration from Cyt c, which triggered heme displacement. The primary dehydration site was estimated to be around surface-exposed hydrophobic residues near the heme center: Ile81 and Val83. These findings and our previous studies, which showed that hydrated water molecules around Ile81 and Val83 are expelled when Cyt c forms a complex with CcO, proposed that dehydration of these residues is functionally significant to electron transfer from Cyt c to CcO.     

Contribution of Protein Conformation to Heme Stereochemistry and Reactivity. Low-Temperature Magnetic Circular Dichroism Data

Abstract

Visible and near infrared magnetic circular dichroism (MCD) spectra of heme proteins and enzymes as well as those of a protein-free heme bound to 2-methylimidazole were recorded and compared at 4.2 K in unrelaxed metastable and relaxed equilibrium heme stereochemistry. The relaxed and unrelaxed stereochemistries of a 5-coordinate ferrous heme were generated by chemical reduction of iron at room temperature before freezing the sample and by photolysis of CO or O₂ complexes at 4.2 K, respectively. The results are discussed in terms of a protein contribution into energies of the Fe-N_epslion(His) and Fe-N(pyrrols) bonds and their change on a ligand binding. We observed and analyzed cases of weak (myoglobin, hemoglobin) and strong (leghemoglobin, peroxidases) constraints imposed by the protein conformation on the proximal heme stereochemistry by comparing the bond energies in proteins with those inthe protoheme-(2-methylimidazole) model compound. The role of a protein moiety in modulating the ligand binding properties of leghemoglobin and the heme reactivity of horseradish peroxidase is discussed.     

Characterization of a novel cytochrome cGJ as the electron acceptor of XoxF-MDH in the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV

Methanotrophs play a prominent role in the global carbon cycle, by oxidizing the potent greenhouse gas methane to CO₂. Methane is first converted into methanol by methane monooxygenase. This methanol is subsequently oxidized by either a calcium-dependent MxaF-type or a lanthanide-dependent XoxF-type methanol dehydrogenase (MDH). Electrons from methanol oxidation are shuttled to a cytochrome redox partner, termed cytochrome c_L. Here, the cytochrome c_L homolog from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV was characterized. SolV cytochrome c_GJ is a fusion of a XoxG cytochrome and a periplasmic binding protein XoxJ. Here we show that XoxGJ functions as the direct electron acceptor of its corresponding XoxF-type MDH and can sustain methanol turnover, when a secondary cytochrome is present as final electron acceptor. SolV cytochrome c_GJ (XoxGJ) further displays a unique, red-shifted absorbance spectrum, with a Soret and Q bands at 440, 553 and 595 nm in the reduced state, respectively. VTVH-MCD spectroscopy revealed the presence of a low spin iron heme and the data further shows that the heme group exhibits minimal ruffling. The midpoint potential E_m,pH7 of +240 mV is similar to other cytochrome c_L type proteins but remarkably, the midpoint potential of cytochrome c_GJ was not influenced by lowering the pH. Cytochrome c_GJ represents the first example of a cytochrome from a strictly lanthanide-dependent methylotrophic microorganism.