Purification of cytochrome b-245 from human neutrophils.

The low potential cytochrome b (b-245) of the microbicidal oxidase of phagocytic cells has been purified from neutrophils from patients with chronic myeloid leukaemia. Cells were homogenized in the presence of proteinase inhibitors and centrifuged to remove the cytoplasm. The pellets containing membranes, granules and other organelles (15 mg/ml) were then washed with buffered sodium cholate (5 mg/ml). Residual pellets were subsequently solubilized with the non-ionic detergent Triton N 101 (10 mg/ml) which extracted about 60% of the cytochrome b. About 10% of the cytochrome b was of mitochondrial origin which was removed on a column of n-amino-octyl-Sepharose that did not adsorb cytochrome b-245. Cytochrome b-245 was chromatographed on a column of heparin-agarose and eluted with NaCl to give a peak specific content of 11-16 nmol of cytochrome b-245/mg of protein, representing a 140-200-fold purification with a recovery of 15%. This technique results in the purification of approx. 100-150 nmol of highly purified cytochrome b-245 from (3-5) X 10(11) cells within 4 days. The most purified material gave a broad band with an apparent Mr of between 68 000 and 78 000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, but gel filtration indicated an aggregated form of the protein in Triton N101 . Purified protein (14 nmol of haem/mg of protein) did not contain FAD or FMN and had no NADPH-dependent O2--generating activity.     

Chloroplast cytochrome b-6. Molecular composition as a lipoprotein.

Disc electrophoretically homogeneous spinach-chloroplast cytochrome b-6 was found to be a lipoprotein whose redox potential was essentially unchanged during isolation. These results further support the hypothesis of Triton X-100/4 M urea, pH 8, as a useful extracting medium for membrane lipoproteins. Cytochrome b-6 was found to have a heme equivalent dry weight of 1 mol of heme per 60000 g. Of this, 20000 g was lipid-extractable. The molecular weight was 60000 with a partial specific volume of 0.84 ml/g. The protein portion of the molecule (40000) consisted of 1 polypeptide chain of 20000 daltons, 1 of 9600 daltons and 2 of 6600 daltons. A simple lipid composition (relative to the original membrane) was found consisting of 7 mol of chlorophyll a and 6 mol of cardiolipin per mol of cytochrome; these two lipids thus account for about 75-80% of the lipid content. An unidentified minor neutral lipid and minor polar lipid were also detected. At pH 7.0 in the presence of 0.5% Triton X-100, E'-o was -0.080 V, and in the absence of Triton X-100, E'-o was -0.120 V. At pH 8 in 0.5% Triton X-100, E'-o was -0.084 V, thus indicating that the redox potential is independent of pH in the region 7-8. The redox reaction proceeded via a one-electron-transfer.     

Light-induced redox changes of cytochrome b-559

Dark incubation of spinach or pea chloroplasts with 10 μm carbonylcyanide m-chlorophenylhydrazone (CCCP) had a negligible effect either on the redox state or the redox potential of the high potential form of cytochrome b-559 (cytochrome b-559_hp). A similar result was obtained with spinach chloroplasts on incubation with 3.3 μm carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), but pea chloroplasts showed a decrease of 10–20% in the amount of reduced cytochrome b-559.Light-induced redox changes of cytochrome b-559 were not observed in untreated spinach chloroplasts. In the presence of CCP or FCCP, cytochrome b-559 was photooxidized both in 655 nm actinic light and in far-red light. Addition of the plastoquinone antagonist, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) to CCCP- or FCCP-treated chloroplasts had only a small effect on the photooxidation of cytochrome b-559 in 655 light, but it completely inhibited the oxidation in far-red light.Electron flow from water to 2,3′,6-trichlorophenolindophenol was partly inhibited by CCCP or FCCP, but the degree of inhibition does not appear to be sufficient to account for the photooxidation of cytochrome b-559.The photooxidation of cytochrome b-559 by 655 nm light at liquid nitrogen temperature was not influenced by prior treatment of the chloroplasts at room temperature with CCCP, DBMIB, or CCCP + DBMIB.The results cannot be explained by the presence of two independent pools of cytochrome b-559 in CCCP-treated chloroplasts, one photooxidized by Photosystem II and the other photooxidized by Photosystem I and photoreduced by Photosystem II.     

The development of cytochrome b-245 in maturing human macrophages.

Cytochrome b-245, the putative terminal component of the specialized cidal oxidase system of phagocytes, was measured in human monocytes in culture. There was a dramatic synthesis of the cytochrome, which increased by 27.3 +/- 2.0 pmol/day per 10(7) cells. This represents an increase of about 40%/day in the early stages and an overall 7-fold increase after 16 days. The protein content increased 3-fold over the same period, resulting in a doubling of the specific content of the cytochrome b. The newly synthesized cytochrome b was identified as that specifically located in the microbicidal oxidase electron-transport chain, as titration demonstrated that, at day 16 of maturation, 70% of the total membrane cytochrome b had a very low midpoint potential (-260 to -220 mV), characteristic of that found in this oxidase system. This cytochrome distributed with the plasma membrane on analytical subcellular fractionation, and a close relationship was observed between the maturation-induced increase in the concentration of this molecule and the capacity of the cells to produce superoxide.     

Purification and properties of cytochrome b-562.5 from Ulva pertusa.

Cytochrome b-562.5 (Ulva pertusa) was extracted from a green alga, U. pertusa, by homogenization of the thalli in phosphate buffer solution. Purification was carried out by acrinol treatment, ammonium sulfate fractionation, DEAE-cellulose and DEAE-Sephadex column chromatographies, and Sephadex gel filtration. Cytochrome b-562.5 has absorption maxima at 562.5 (alpha), 530.5 (beta), 429 (gamma), and 326 nm (delta) in the reduced form and at 537, 415 (gamma), and 275 nm in the oxidized form. The alpha-band of the reduced form is asymmetric with a shoulder at 560 nm, at liquid nitrogen temperature this band splits into two distinct peaks at 562 and 556.5 nm. The absorption maxima of the pyridine ferrohemochrome appear at 556 (alpha), 523 (beta), and 418 nm (gamma). The cytochrome does not combine with carbon monoxide or cyanide. The preparation of the cytochrome shows little peroxidase activity. The cytochrome is oxidized by ferricyanide and reduced by cysteine, ascorbate, and hydrosulfite. Autoxidation of the cytochrome was found to be very slow. The midpoint potential (Em) of the cytochrome was determined by equilibration with the ferro- and ferri-EDTA system to be +0.20 V at pH7.0. The molecular weight of the cytochrome was estimated by Sephadex gel filtration to be 23x10(3).     

The many roles of cytochrome b-5 in hepatic microsomes.

The purpose of this report is to review the current literature on cytochrome $\text{b}$₅ in hepatic microsomes and to draw conclusions as to its role in microsomal electron transfer pathways. For details concerning the history of cytochrome $\text{b}$₅ the reader is reffered to reviews by C. F. Strittmatter (1) and P. Strittmatter (2). For information on the chemistry of cytochrome $\text{b}$₅ the reader is reffered to the papers by Ozols and Strittmatter (3), Kajihara and Hagihara (4), and Ehrenberg and Bois-Poltoratsky (5). For more recent studies on the isolation and properties of detergent solubilized cytochrome $\text{b}$₅, which contains a hydrophobic peptide enabling reincorporation into membranes, the reader is referred to references 6-12.For simplicity, this minireview is divided into four parts, reflecting areas of study on the role of cytochrome $\text{b}$₅ in the microsomes. One major area is in fatty acid 9 desaturation. Two other areas concern cytochrome $\text{b}$₅ involvement in cytochrome P-450 mediated mixed function oxidations. The fourth section deals with other non-cytochrome P-450 pathways in which cytochrome $\text{b}$₅ is suggested as being a component.     

The enigmatic cytochrome b-559 of oxygenic photosynthesis

The ubiquitous and obligatory association of cytochrome b -559 with the photosystem II reaction center of oxygenic photosynthesis is a conundrum since it seems not to have a function in the primary electron transport pathway of oxygen evolution. A model for the cytochrome structure that satisfies the cis -positive rule for membrane protein assembly consists of two short, non-identical hydrophobic membrane-spanning polypeptides (α and β), each containing a single histidine residue, as ligands for the bridging heme prosthetic group that is on the side of the membrane opposite to the water splitting apparatus. The ability of the heterodimer, but not the single α-subunit, to satisfy the cis -positive rule implies that the cytochrome inserts into the membrane as a heterodimer, with some evidence implicating it as the first membrane inserted unit of the assembling reaction center. The very positive redox potential of the cytochrome can be explained by a position for the heme in a hydrophobic niche near the stromal aqueous interface where it is also influenced by the large positive dipole potential of the parallel α-helices of the cytochrome. The requirement for the cytochrome in oxygenic photosynthesis may be a consequence of the presence of the strongly oxidizing reaction center needed for H₂O-splitting. This may lead to the need, under conditions of stress or plastid development, for an alternate source of electrons when the H₂O-splitting system is not operative as a source of reductant for the reaction center.     

Kinetic studies of the reduction of neutrophil cytochrome b-558 by dithionite.

The reduction with dithionite of neutrophil cytochrome b-558, implicated in superoxide generation by activated neutrophils, was investigated by a stopped-flow technique in non-ionic-detergent extracts of the membranes and in crude membrane particles. The dependence of the pseudo-first-order rate constants on the concentration of dithionite was consistent with a mechanism of reduction that involves the dithionite anion monomer SO2.- as the reactive species. The estimated second-order rate constant was 7.8 X 10(6) M-1 X S-1 for Lubrol PX-solubilized cytochrome b-558 and 5.1 X 10(6) M-1 X S-1 for the membrane-bound protein. The similarity of the kinetic constants suggests that solubilization did not introduce gross changes in the reactive site. Imidazole and p-chloromercuribenzoate, known as inhibitors of NADPH oxidase, did not affect significantly cytochrome b-558 reduction rates. The reaction rate of cytochrome b-558 with dithionite exhibited a near-zero activation energy. The first-order rate constant for reduction decreased with increasing ionic strength, indicating a positive effective charge on the reacting protein.     

Several forms of chromaffin granule cytochrome b-561 revealed by EPR spectroscopy.

Low-temperature EPR spectra of chromaffin granule membranes from bovine adrenal medulla reveal 3 different signals of the ferric cytochrome b-561. A typical gZ signal of a low-spin cytochrome observed at g approximately 3 is comprised of a high-potential component with gZ = 3.14 and a low-potential one with gZ = 3.11, the low-potential signal showing significantly faster relaxation. In addition, a highly temperature-sensitive heme signal at g = 3.7 is observed which is fully retained in the preparation of granule membranes with b-561 reduced by 50% but disappears upon full reduction of the cytochrome by ascorbate. The signal is strikingly similar to that of the mitochondrial low-potential cytochrome b heme (bL or b-566). The presence of several forms of b-561 in chromaffin granule membranes may provide a structural basis for the transmembrane electron transfer believe to be catalyzed by this hemoprotein.     

Concerning the enigmatic cytochrome b-559 of oxygenic photosynthesis

Photosynthesis Research - Although there is an extensive literature on the properties and possible electron transfer pathways of cytochrome b-559, which is a prominent subunit of the multi-subunit...     

On the structure and function of cytochrome b-559

A sumary of biochemical, biophysical, and molecular biological data is presented which led to the identification of two different polypeptides ( and , MW=9.16 and 4.27 kDa) in the cytochrome b-559 protein. The presence of a single His residue on each polypeptide, and the conclusion from spectroscopy that the heme coordination must be bis-histidine led to an obligatory requirement for coordination of a single heme through a heme cross-linked dimer. This structure does not have a precedent among soluble or membrane bound cytochromes. The possible participation of the cytochrome in the pathway of photoactivation is discussed.     

The enzymic reduction and kinetics of oxidation of cytochrome b-245 of neutrophils.

1. The absorption coefficient of human neutrophil plasma-membrane reduced-minus-oxidized cytochrome b-245 was determined [delta epsilon (mM; 559-540 nm) = 21.6 cm-1]. 2. Neutrophil polymorphonuclear leucocytes (neutrophils) were prepared from human, ox, horse and pig blood. In each case plasma-membrane fractions were found to contain low-potential cytochrome b. When membranes from horse neutrophils were incubated anaerobically with either NADH or NADPH the cytochrome b became reduced. Prior stimulation of the cells with phorbol myristate acetate did not increase the rate or extent of cytochrome b reduction in isolated membranes, but did increase both the rate and extent of reduction by NADPH in Triton-treated cells. 3. A cytochrome b was present also in the specific granule fraction of human neutrophils. Its Em (pH 7.0) was found to be -248 mV, very similar to that of the plasma-membrane cytochrome b. 4. The rate of oxidation of reduce cytochrome b-245 by air-saturated buffer, was determined by using stopped-flow techniques. In intact membranes t 1/2 for oxidation was 4.7 ms. This rate is sufficiently rapid to support the view that cytochrome b-245 is the oxidase in the respiratory burst of neutrophils. 5. Plasma-membrane cytochrome b of human neutrophils formed a complex with CO. At room temperature and 1 atm of CO approx. 40% of the cytochrome formed a complex; approx. 60% binding was measured at the increased concentration of dissolved CO achieved at 5 degrees C. The concentration of CO giving 50% binding was 1.18 mM.     

Kinetics of the photoreduction of cytochrome b-559 by photosystem II in chloroplasts.

The kinetics of the photoreduction of cytochrome b-559 and plastoquinone were measured using well-coupled spinach chloroplasts. High potential (i.e, hydroquinone reducible) cytochrome b-559 was oxidized with low intensity far-red light in the presence of N-methyl phenazonium methosulfate or after preillumination with high intensity light. Using long flashes of red light, the half-reduction time of cytochrome b-559 was found to be 100 +/- 10 ms, compared to 6-10 ms for the photoreduction of the plastoquinone pool. Light saturation of the photoreduction of cytochrome b-559 occurred at a light intensity less than one-third of the intensity necessary for the saturation of ferricyanide reduction under identical illumination conditions. The photoreduction of cytochrome b-559 was accelerated in the presence of dibromothymoquinone with a t 1/2 = 25-35 ms. The addition of uncouplers, which caused stimulatory effect on ferricyanide reduction under the same experimental conditions resulted in a decrease in the rate of cytochrome b-559 reduction. The relatively slow photoreduction rate of cytochrome b-559 compared to the plastoquinone pool implies that electrons can be transferred efficiently from Photosystem II to plastoquinone without the involvement of cytochrome b-559 as an intermediate. These results indicate that it is unlikely that high potential cytochrome b-559 functions as an obligatory redox component in the main electron transport chain joining the two photosystems.     

The amino acid sequence of cytochrome c-555 from the methane-oxidizing bacterium Methylococcus capsulatus.

The amino acid sequence of the cytochrome c-555 from the obligate methanotroph Methylococcus capsulatus strain Bath (N.C.I.B. 11132) was determined. It is a single polypeptide chain of 96 residues, binding a haem group through the cysteine residues at positions 19 and 22, and the only methionine residue is a position 59. The sequence does not closely resemble that of any other cytochrome c that has yet been characterized. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50131 (12 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment.     

Light-induced turnover of chloroplast cytochrome b-559 in the presence of N-methylphenazonium methosulphate.

In the presence of 0.1-5 muM N-methylphenazonium methosulphate approx. 50-70% oxidation of cytochrome b-559 can be induced by far-red light. The oxidation is best observed with long wavelength far-red light (732 nm) of moderate intensities (approx. 10(4) ergs/cm2 per s) and is reversed by subsequent illumination with red light. Concentrations of N-methylphenazonium methosulphate above 5 muM are inhibitory probably due to cyclic electron flow. The far-red oxidation is inhibited by low concentrations of the plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, while 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibits red light reduction and increases the amplitude of far-red oxidation. The effect of N-methylphenazonium methosulphate is mimicked by N-methyl-phenazonium ethosulphate, but not by pyocyanine or diaminodurene. Low concentrations (2-3 muM) of N-methylphenazonium methosulphate also stimulate a 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-inhibitable red light reduction of cytochrome f.