Effects of N,N′-dicyclohexylcarbodiimide on isolated and reconstituted cytochrome b-c1 complex from bovine heart mitochondria

N,N′-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstitued mitochondrial cytochrome b-c₁ complex. DCCD inhibits equally electron flow and proton translocation (i.e., the $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratio is not affected) catalysed by the enzyme reconstituted into phospholipid vesicles. The inhibitory effects are accompanied by structural alterations in the polypeptide pattern of both isolated and reconstituted enzyme. Cross-linking was observed between subunits V (iron-sulfur protein) and VII, indicating that these polypeptides are in close proximity. A clear correlation was found between the kinetics of inhibition of enzymic activity and the cross-linking, suggesting that the two phenomena may be coupled. Binding of [¹⁴C]DCCD was also observed, to all subunits with the isolated enzyme and preferentially to cytochrome b with the reconstituted vesicles; in both cases, however, it was not correlated kinetically with the inhibition of the enzymic activity.     

Reconstitution of b-type cytochrome oxidase from Rhodopseudomonas capsulata in liposomes and turnover studies of proton translocation

Purified b-type cytochrome oxidase from Rhodopseudomonas capsulata was incorporated into phospholipid vesicles to measure proton extrusion with pulses of ferrocytochrome c for one oxidase turnover. In accordance with the pH shift of its midpoint potential, the purified oxidase showed a proton extrusion of 0.24 $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ with uptake of 1 $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ from the liposomes for the reduction of oxygen to water. This proton translocation could only be observed in the presence of valinomycin +K⁺ and was not inhibited by DCCD. Oxidase preparations from the first purification step, which contain other protein compounds especially a membrane-bound cytochrome c but not the ubiquinol-cytochrome c₂-oxidoreductase showed a pumping activity of 0.9 $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$, which was inhibited by DCCD for nearly 75%. Inhibition of the electron transfer was not observed, which could be explained by a ‘molecular slipping’ of proton extrusion and electron transfer. Proton extrusion from two oxidase-turnovers was only 80% of that from one turnover. The proton pumping of the b-type oxidase strongly depended on the enzyme/phospholipid ratio.     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Electron transfer by b- and c-type cytochromes in relation to the origin of the ‘slow’ electric potential component

Cytochrome redox changes and electric potential generation are kinetically compared during cyclic electron transfer in Photosystem-I-enriched and Photosystem-II-depleted subchloroplast vesicles (i.e., stroma lamellae membrane vesicles) supplemented with ferredoxin using a suitable electron donating system. In response to a single-turnover flash, the sequence of events is: (1) fast reduction of cytochrome b-563 (t_0.5 ≈ 0.5 ms) (2) oxidation of cytochrome c-554 (t_0.5 ≈ 2 ms), (3) slower reduction of cytochrome b-563 (t_0.5 ≈ 4 ms), (4) generation of the ‘slow’ electric potential component (t_0.5 ≈ 15–20 ms), (5) re-reduction of cytochrome c-554 (t_0.5 ≈ 30 ms) and (6) reoxidation of cytochrome b-563t_0.5 ≈ 90 ms). Per flash two cytochrome b-563 species turn over for one cytochrome c-554. These b-563 cytochromes are reduced with different kinetics via different pathways. The fast reductive pathway proceeds probably via ferredoxin, is insensitive to DNP-INT, DBMIB and HQNO and is independent on the dark redox state of the electron transfer chain. In contrast, the slow reductive pathway is sensitive to DNP-INT and DBMIB, is strongly delayed at suboptimal redox poising (i.e., low $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ ratio) and is possibly coupled to the reduction of cytochrome c-554. Each reductive pathway seems obligatory for the generation of about 50% of the slow electric potential component. Also cytochrome c-559_LP (LP, low potential) is involved in Photosystem-I-associated cyclic electron flow, but its flash-induced turnover is only observed at low preestablished electron pressure on the electron-transfer chain. Data suggest that cyclic electron flow around Photosystem I only proceeds if cytochrome b-559_LP is in the reduced state before the flash, and a tentative model is presented for electron transfer through the cyclic system.     

Catalytic properties of the resolved flavoprotein and cytochrome B components of the NADPH dependent O2−• generating oxidase from human neutrophils

The resolved flavoprotein and cytochrome b₅₅₉ components of the NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generating oxidase from human neutrophils were the subject of further study. The resolved flavoprotein, depleted of cytochrome b₅₅₉, was reduced by NADPH under anaerobic conditions and reoxidized by oxygen. NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generation by the resolved flavoprotein fraction was not detectable, however it was competent in the transfer of electrons from NADPH to artificial electron acceptors. The resolved cytochrome b₅₅₉, depleted of flavoprotein, demonstrated no measureable NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generating activity and was not reduced by NADPH under anaerobic conditions. The dithionite reduced form of the resolved cytochrome b₅₅₉ was rapidly oxidized by oxygen, as was the cytochrome b₅₅₉ in the intact oxidase.     

Ubisemiquinone radicals from the cytochrome b-c1 complex of the mitochondrial electron transport chain--demonstration of QP-S radical formation

Stable ubisemiquinone radical(s) in the cytochrome $\text{b}\text{?}\text{c}{}_1$-II complex of bovine heart was observed following reduction by succinate in the presence of catalytic amounts of succinate dehydrogenase. The radical was abolished by addition of antimycin A, but a residual radical remained in the presence of excess exogenous Q₂. The radical showed an EPR signal of g = 2.0046 ± .003 at X band (～9.4 GHz) with no resolved hyperfine structure and had a line width of 8.1 ± .5 Gauss at 23°C. The Q band (35 GHz) spectra showed wellresolved $\text{g}$-anisotropy and had a field separation between derivative extrema of 26 ± 1 Gauss. This radical is evidently from QP-C. These observations substantiate that the radical is immobilized and bound to a protein. The QP-S radical was demonstrated in the cytochrome $\text{b}\text{-}\text{c}{}_1$-II complex only in the presence of more than a catalytic amount of succinate dehydrogenase and cytochrome $\text{b}\text{-}\text{c}{}_1$. This signal was not antimycin a inhibitory. The signal amplitude paralleled the reconstitutive enzymic activity of succinate-cytochrome $\text{c}$ reductase from succinate dehydrogenase and the cytochrome $\text{b}\text{-}\text{c}{}_1$-II complex.     

Effect of trifluoperazine on skeletal muscle mitochondrial respiration

The effect of trifluoperazine on the respiration of porcine liver and skeletal muscle mitochondria was investigated by polarographic and spectroscopic techniques. Low concentrations of trifluoperazine (88 nmol/mg protein) inhibited both the ADP- and Ca²⁺-stimulated oxidation of succinate, and reduced the values of the respiratory control index and the $\text{ADP}\text{O}$ and $\text{Ca}{}^{\mathrm{2+}}\text{O}$ ratio. High concentrations inhibited both succinate and ascorbate plus tetramethyl-p-phenylenediame (TMPD) oxidations, and uncoupler (carbonyl cyanide p-trifluromethoxyphenylhydrazone) and Ca²⁺-stimulated respiration. Porcine liver mitochondria were more sensitive to trifluoperazine than skeletal muscle mitochondria. Trifluoperazine inhibited the electron transport of succinate oxidation of skeletal muscle mitochondria within the cytochrome b-c₁ and cytochrome c₁-aa₃ segments of the respiratory chain system. 233 nmol trifluoperazine/mg protein inhibited the aerobic steady-state reduction of cytochrome c₁ by 92% with succinate as substrate, and of cytochrome c and cytochrome aa₃ by 50–60% with ascorbate plus TMPD as electron donors. Trifluoperazine can thus inhibit calmodulin-independent reactions particularly when used at high concentrations.     

Evidence for a protonmotive Q cycle mechanism of electron transfer through the cytochrome b-c1 complex.

A protein named oxidation factor can be reversibly removed from succinate-cytochrome $\text{c}$ reductase complex and shown to be required for electron transfer between succinate and cytochrome $\text{c}$. This protein is required for reduction of cytochrome $\text{c}{}_1$ and, in the presence of antimycin, for reduction of both cytochromes $\text{b}$ and $\text{c}{}_1$. These results are consistent with a protonmotive Q cycle mechanism in which the oxidation factor catalyzes electron transfer from reduced quinone to cytochrome $\text{c}{}_1$ and thus liberates from reduced quinone one of two protons required for energy conservation during electron transfer through the cytochrome $\text{b}\text{-}\text{c}{}_1$ complex.     

The concept of high- and low-affinity reactions in bovine cytochrome c oxidase steady-state kinetics

(1) Analysis of the data from steady-state kinetic studies shows that two reactions between cytochrome c and cytochrome c oxidase sufficed to describe the concave Eadie-Hofstee plots ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{? 1 · 10}{}^{\mathrm{?8}}\text{M}$ and $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{? 2 · 10}{}^{\mathrm{?5}}\text{M}$). It is not necessary to postulate a third reaction of $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{? 10}{}^{\mathrm{?6}}\text{M}$. (2) Change of temperature, type of detergent and type of cytochrome c affected both reactions to the same extent. The presence of only a single catalytic cytochrome c interaction site on the oxidase could explain the kinetic data. (3) Our experiments support the notion that, at least under our conditions (pH 7.8, low-ionic strength), the dissociation of ferricytochrome c from cytochrome c oxidase is the rate-limiting step in the steady-state kinetics. (4) A series of models, proposed to describe the observed steady-state kinetics, is discussed.     

Oxidation of anionic nitroalkanes by flavoenzymes,and participation of superoxide anion in the catalysis

The reactivities of anionic nitroalkanes with 2-nitropropane dioxygenase of Hansenula mrakii, glucose oxidase of Aspergillus niger, and mammalian d-amino acid oxidase have been compared kinetically. 2-Nitropropane dioxygenase is 1200 and 4800 times more active with anionic 2-nitropropane than d-amino acid oxidase and glucose oxidase, respectively. The apparent K_m values for anionic 2-nitropropane are as follows: 2-nitropropane dioxygenase, 1.61 mm; glucose oxidase, 16.7 mm; and d-amino acid oxidase, 11.1 mm. Anionic 2-nitropropane undergoes an oxygenase reaction with 2-nitropropane dioxygenase and glucose oxidase, and an oxidase reaction with d-amino acid oxidase. In contrast, anionic nitroethane is oxidized through an oxygenase reaction by 2-nitropropane dioxygenase, and through an oxidase reaction by glucose oxidase. All nitroalkane oxidations by these three flavoenzymes are inhibited by Cu and Zn-superoxide dismutase of bovine blood, Mn-superoxide dismutases of bacilli, Fe-superoxide dismutase of Serratia marcescens, and other $\text{O}{}_2{}^{\mathrm{?}}$ scavengers such as cytochrome c and NADH, but are not affected by hydroxyl radical scavengers such as mannitol. None of the $\text{O}{}_2{}^{\mathrm{?}}$ scavengers tested affected the inherent substrate oxidation by glucose oxidase and d-amino acid oxidase. Furthermore, the generation of $\text{O}{}_2{}^{\mathrm{?}}$ in the oxidation of anionic 2-nitropropane by 2-nitropropane dioxygenase was revealed by ESR spectroscoy. The ESR spectrum of anionic 2-nitropropane plus 2-nitropropane dioxygenase shows signals at g₁ = 2.007 and g₁₁ = 2.051, which are characteristic of $\text{O}{}_2{}^{\mathrm{?}}$. The $\text{O}{}_2{}^{\mathrm{?}}$ generated is a catalytically essential intermediate in the oxidation of anionic nitroalkanes by the enzymes.     

Cytochrome b5 as electron donor to rabbit liver cytochrome P-450LM2 in reconstituted phospholipid vesicles

When incorporated into phospholipid vesicles containing NADPH-cytochrome P-450 reductase and P-450_LM2, cytochrome b₅ enhanced the rate of NADPH-supported hydroxylation of 7-ethoxycoumarin or $\text{p}$-nitroanisole about 5-fold. Cytochrome b₅ did not affect the rate of NADPH-oxidation, nor the rate of NADPH-supported formation of the ferrous CO-complex of cytochrome P-450. However, the cytochrome b₅-mediated increase in product formation was found to be correlated with concomitant decreases in the production of H₂O₂ or $\text{O}{}_2{}^{\mathrm{?}}$ in the system, thus strongly indicating cytochrome b₅ being a more efficient donor of the second electron to cytochrome P-450 than is NADPH-cytochrome P-450 reductase.     

Studies on the mechanism of electron transport in the bc1-segment of the respiratory chain in yeast. II. The binding of antimycin to mitochondrial particles and the function of two different binding sites

1. In mitochondrial particles antimycin binds to two separate specific sites with dissociation constants $\text{K}{}_{\mathrm{<mtext>d</mtext><mtext>1</mtext>}}\text{≦ 4 · 10}{}^{\mathrm{?13}}\text{M}$ and $\text{K}{}_{\mathrm{<mtext>d</mtext><mtext>2</mtext>}}\text{= 3 · 10}{}^{\mathrm{?9}}\text{M}$, respectively.2. The concentrations of the two antimycin binding sites are about equal. The absolute concentration for each binding site is about 100 – 150 pmol per mg of mitochondrial protein.3. Antimycin bound to the stronger site mainly inhibits NADH- and succinate oxidase. Binding of antimycin to the weaker binding site inhibits the electron flux to exogenously added cytochrome c after blocking cytochrome oxidase by KCN.4. Under certain conditions cytochrome b and c₁ are dispensible components for antimycin-sensitive electron transport.5. A model of the respiratory chain in yeast is proposed which accounts for the results reported here and previously. (Lang, B., Burger, G. and Bandlow, W. (1974) Biochim. Biophys. Acta 368, 71–85).     

Proton pump coupled to cytochrome c oxidase in Paracoccus denitrificans

The proton translocating properties of cytochrome c oxidase in whole cells of Paracoccus denitrificans have been studied with the oxidant pulse method.$\text{→}\text{H}{}^{\mathrm{+}}\text{2e}{}^{\mathrm{?}}$ quotients have been measured with endogenous substrates, added methanol and added ascorbate (+TMPD) as reductants, and oxygen and ferricyanide as oxidants. It was found that both the observed $\text{→}\text{H}{}^{\mathrm{+}}\text{O}$ with ascorbate (+TMPD) as reductant, and the differences in proton ejection between oxygenand ferricyanide pulses, with endogenous substrates or added methanol as a substrte, indicate that the P. denitrificans cytochrome c oxidase translocates protons with a stoichiometry of $\text{2}\text{H}{}^{\mathrm{+}}\text{2e}{}^{\mathrm{?}}$. The results presented in this and previous papers are in good agreement with recent findings concerning the mitochondrial cytochrome c oxidase, and suggest unequal charge separation by different coupling segments of the respiratory chain of P. denitrificans.     

Activation of heart sarcolemmal Ca2+/Mg2+ ATPase by cyclic AMP-dependent protein kinase.

The sarcolemmal membrane obtained from rat heart by hypotonic shock-LiBr treatment method was found to incorporate ³²P from [γ-³²P] ATP in the absence and presence of cyclic AMP and protein kinase. The phosphorylated membrane showed an increase in Ca²⁺ ATPase and Mg²⁺ ATPase activities without any changes in Na⁺K⁺ ATPase activity. The observed increase in $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase activity was found to be associated with an increase in V_max value of the reaction whereas K_a value for $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ was not altered. These results provide information concerning biochemical mechanism for increased calcium entry due to hormones which are known to elevate cyclic AMP levels in myocardium and produce a positive inotropic effect.     

Kinetic and thermodynamic properties of membrane-bound cytochromes of aerobically and photosynthetically grown Rhodopseudomonas spheroides

A green mutant of Rhodopseudomonas spheroides was isolated in which spectroscopic measurements of the α-band region of cytochromes could be made. It was grown either aerobically or photosynthetically, and the membrane fractions prepared from cells of each type. Anaerobic potentiometric titration at 560 nm minus 542 nm showed the same three redox components, tentatively identified as b-type cytochromes, in membrane fractions from either type of cell. The mid-point potentials were approximately +185, +41 and ?104 mV. In membranes from photosynthetically grown cells the major cytochrome form absorbing at 560 nm had a mid-point potential of +42 mV; in aerobically grown cells the major form had a potential of +185 mV. In both types of cell only one c-type cytochrome was found, with a mid-point potential of +295 mV. An a-type cytochrome was present only in aerobically-grown cells.Substrate-reduced particles from these cells were mixed with air-saturated buffer in a stopped-flow spectrophotometer and the kinetics of oxidation of b- and c-type cytochromes were measured. The same two b-type components, reacting with pseudo first order kinetics, were detected in particles from both aerobically and photosynthetically grown cells ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for oxidation 1.3 s and 0.13 s). The c-type cytochrome of particles from aerobically grown cells was oxidised with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.97 s; the c-type cytochrome of photosynthetic cells was oxidised faster, with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.27 s.These observations have implications on the adaptive formation of electron transport systems that are discussed.     

The insect brain (Na+ + K+)-ATPase Binding of ouabain in the hawk moth, Manduca sexta

(1) A quantitative study has been made of the binding of ouabain to the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ will bind ouabain either in the presence of Mg²⁺ and P_i, (‘Mg²⁺, P_i’ conditions) or in the presence of Na⁺, Mg²⁺, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$) of ouabain to the M. sexta brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ under both binding conditions. However, ouabain binding is more sensitive to K⁺ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K⁺ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has a higher $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ can bind ouabain.     

The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain

1. Formate inhibits cytochrome $\text{c}$ oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome $\text{aa}{}_3$. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome $\text{aa}{}_3\text{(a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}$) and in the half-reduced species ($\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}$). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the $\text{aa}{}_3$ spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of $\text{a}{}_3{}^{\mathrm{2+}}$ minus $\text{a}{}_3{}^{\mathrm{3+}}$, free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome $\text{a}{}_3{}^{\mathrm{3+}}$) and azide (which induces peak shifts of cytochrome $\text{a}{}^{\mathrm{2+}}$ towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome $\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$ is faster than its rate of dissociation from $\text{a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$, especially in the presence of cytochrome $\text{c}$. The $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome $\text{c}$ reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]².5. Formate inhibition of ascorbate plus $\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenyl-enediamine}$ oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome $\text{c}$ reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome $\text{aa}{}_3$ level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.     

Flash-induced photophosphorylation in Rhodospirillum rubrum chromatophores I. The relationship between cytochrome c-420 content and photophosphorylation

The content of cytochrome c-420 in Rhodospirillum rubrum chromatophores prepared by grinding with alumina is 5–10% of that in whole cells, and 20–40% in chromatophores by ‘French’ pressing.Flash-induced phosphorylation of various chromatophores which varied in cytochrome content from 7 to 40% is proportional to the cytochrome content. Extrapolating the cytochrome c-420 content to that observed in whole cells, a ratio $\text{ATP}\text{P}{}^{\mathrm{+}}\text{X}{}^{\mathrm{?}}$ near 1 is calculated. At low flash intensity the phosphorylation per flash is proportional to flash energy.Photophosphorylation in flashes given after a time of several minutes is only slightly dependent on the number of flashes. If the flashes are spaced from 0.1 to 10 s, relative phosphorylation in the first flash is about 70% and in the second 90% of that observed in the following flashes. Proton binding is not affected by the cytochrome c-420 content and a ratio of $\text{H}{}^{\mathrm{+}}\text{P}{}^{\mathrm{+}}\text{X}{}^{\mathrm{?}}$ of 2.3 was found.These results can be explained by a working hypothesis in which charge separation occurring at one reaction centre and the resulting electron transport mediated amongst others by c-420, results in the injection of two protons into an ATPase, this in contrast to a chemiosmotic mechanism, where the protons are released in the chromatophore inner space.     

Chemiluminescence in the reaction of cytochrome c with hydrogen peroxide

(1) Aqueous solutions of 1–10 μM ferricytochrome c treated with 100 μM–100 mM H₂O₂ at pH 8.0 emit chemiluminescence with quantum yield $\text{Ф ? 10}{}^{\mathrm{?9}}$ and absolute maximum intensity $\text{I}{}_{\mathrm{<mtext>max</mtext>}}\text{? 10}{}^5\text{hv/}\text{s}$ per cm³ (λ = 440), and exhibit exponential decay with a rate constant of 0.15 s^?1. (2) The emission spectrum of the chemiluminescence covers the range 380–620 nm with the maximum at 460 ± 10 nm. (3) Neither cytochrome c nor haemin fluoresce in the spectral region of the chemiluminescence. In the reaction course with H₂O₂, a weak fluorescence in the region 400–620 nm with λ_max = 465–510 nm (λ_exc 315–430 nm) gradually arises. This originates from tryptophan oxidation products of the formylkynurenine type or from imidazole derivatives, respectively. (4) Frozen solutions (77 K) of cytochrome c exhibit phosphorescence typical of tryptophan (λ_exc = 280 nm, λ_em = 450 nm). During the peroxidation, an additional phosphorescence gradually appears in the range 480–620 nm with λ_max = 530 nm (λ_exc = 340 nm). This originates from oxidative degradation products of tryptophan. (5) There are no red bands in the chemiluminescence spectra of cytochrome c or haemin. This result suggests that singlet molecular oxygen $\text{O}{}_2\text{(}{}^1\text{Δ}{}_{\mathrm{<mtext>g</mtext>}}\text{)}$ is not involved in either peroxidation or chemiluminescence. (6) The haem Fe³⁺ group and H₂O₂ appear to be crucial for the chemiluminescence. It is suggested that the generation of electronically excited, light-emitting states is coupled to the production of conformational out-of-equilibrium states of peroxy-Fe-protoporphyrin IX compounds.