Characterization of cytochrome b in the isolated ubiquinol-cytochrome c2 oxidoreductase from Rhodopseudomonas sphaeroides GA

Extinction coefficients for cytochrome b and c₁ in the isolated cytochrome bc₁ complex from Rhodopseudomonas sphaeroides GA have been determined. They are 25 mM^?1.cm^?1 at 561 nm for cytochrome b and 17.4 mM^?1.cm^?1 at 553 nM for cytochrome c₁ for the difference between the reduced and the oxidized state. Cytochrome b is present in two forms in the complex. One form has an E_m7 of 50 mV, an α-peak of 557 nm at liquid N₂ temperature and of 561 nm at RT, which is red-shifted by antimycin A. The other form has an E_m7 of ?90 mV, a double α-peak of 555 and 561 nm at liquid N₂ temperature corresponding to 559 and 566 nm at RT. The absorption at 566 nm is red-shifted by myxothiazol. The two shifts are independent of each other. Both midpoint potentials of cytochromes b are pH-dependent. The redox center compositions of the cytochrome bc₁ complexes from Rhodopseudomonas sphaeroides and from mitochondria are identical.     

Purification and partial characterization of cytochrome b5 from Tetrahymena pyriformis

With the use of detergents and successive column chromatographies, Tetrahymena b-type cytochrome was purified from microsomes to a specific content of 36.0 nmol per mg of protein. The purified form showed a single band on SDS-polyacrylamide gel with molecular weight of 22,000. The spectral properties of the reduced b-type cytochrome, the α-peak of which is situated at 560 nm and asymmetric with a shoulder at 556 nm, was different from that of rat liver microsomal cytochrome b₅. However, it was reducible by NADH in the presence of NADH-cytochrome b₅ reductase purified from rat liver microsomes.The results indicated that the microsomal b-type cytochrome should be designated as cytochrome b₅ of a ciliated protozoan, Tetrahymena pyriformis.     

Cytochrome distribution across chloroplast thylakoid membranes. Controlled proteolysis of inside-out and right-side-out vesicles

The transverse distribution of chloroplast cytochromes b-559 (high and low potentials), b-563 and f in pea thylakoid membranes was studied by the effects of trypsin and pronase on inside-out and right-side-out thylakoid vesicles. The high potential (HP) form of cytochrome b-559 was degraded to a low potential (LP) form most rapidly in right-side-out vesicles. In either type of vesicle there was no overall loss of the cytochrome from the membrane. This suggests that the haem group is buried in the membrane but that the cytochrome environment is most labile at the outer surface. Cytochrome b-563 was unaffected by trypsin and only slightly degraded by pronase in inverted vesicles. However, pronase caused the loss of an M_r 1000, non-haem fraction from the cytochrome f polypeptide in inside-out vesices only. The total cytochrome f content (measured spectrophotometrically and by staining polyacrylamide gels for haem associated peroxidase activity) decayed only slightly in either type of vesicle. These observations suggest that cytochrome f is, in part, exposed to the intrathylakoid lumen, whilst its haem group is retained in a more hydrophobic region.     

A method for in situ characterization of b- and c-type cytochromes in Escherichia coli and in Complex III from beef heart mitochondria by combined spectrum deconvolution and potentiometric analysis

An analytical technique for the in situ characterization of b- and c-type cytochromes has been developed. From evaluation of the results of potentiometric measurements and spectrum deconvolutions, it was concluded that an integrated best-fit analysis of potentiometric and spectral data gave the most reliable results. In the total cytochrome b content of cytoplasmic membranes from aerobically grown Escherichia coli, four major components are distinguished with α-band maxima at 77 K of 555.7, 556.7, 558.6 and 563.5 nm, and midpoint potentials at pH 7.0 of 46, 174, ?75 and 187 mV, respectively. In addition, two very small contributions to the α-band spectrum at 547.0 and 560.2 nm, with midpoint potentials of 71 and 169 mV, respectively, have been distinguished. On the basis of their spectral properties they should be designated as a cytochrome c and a cytochrome b, respectively. In Complex III, isolated from beef heart mitochondria, five cytochromes are distinguished: cytochrome c₁ (Λ_m(25°C) = 553.5 nm; E′₀ = 238 mV) and four cytochromes bΛ_m(25°C) = 558.6, 561.2, 562.1, 566.1 nm and E′₀ = ?83, 26, 85, ?60 mV).     

The chloroplast cytochrome b 6 f complex can exist in monomeric and dimeric states

The cytochrome b ₆ f complex isolated from spinach chloroplast membranes can be resolved into two forms, a monomeric and a dimeric form, by centrifugation on sucrose gradients. The conversion of the dimeric form of the complex into the monomeric form could be prevented by cross-linking with the homobifunctional reagent, dithiobis(succinimidylpropionate) but not by cross-linking with disuccinimidyltartrate or glutaraldehyde. SDS-PAGE analyses of the monomeric and dimeric forms of the cytochrome complex showed the presence of specific cross-linked products in each respective form of the complex. For example, the monomeric form contained a cross-linked product of cytochrome f, cytochrome b ₆ f and subunit IV while the dimeric form contained a cross-linked dimer of cytochrome b ₆ f. The presence of the former in the isolated cytochrome b ₆ f complex prepared by the method of Hurt and Hauska (Eur J Biochem 117: 591–599, 1981) indicates the presence of the monomer in his preparation.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DSP dithiobis(succinimidylpropionate) - DST disuccinimidyltartrate     

Involvement of plastoquinone bound within the isolated cytochrome b6-f complex from chloroplasts in oxidant-induced reduction of cytochrome b6

(1) Oxidant-induced reduction of cytochrome b₆ is completely dependent on a reduced component within the isolated cytochrome b₆-f complex. This component can be reduced by dithionite or by NADH/N-methylphenazonium methosulfate. It is a 2H⁺/2e⁻ carrier with a midpoint potential of 100 mV at pH 7.0, which is very similar to the midpoint potential of the plastoquinone pool in chloroplasts. (2) Oxidant-induced reduction of cytochrome b₆ is stimulated by plastoquinol-1 as well as by plastoquinol-9. The midpoint potential of the transient reduction of cytochrome b₆, however, was not shifted by added plastoquinol. (3) Quinone analysis of the purified cytochrome b₆-f complex revealed about one plastoquinone per cytochrome f. The endogenous quinone is heterogeneous, a form more polar than plastoquinone-A, probably plastoquinone-C, dominating, This is different from the thylakoid membrane where plastoquinone-A is the main quinone. (4) The endogenous quinone can be extracted from the lyophilized cytochrome b₆-f complex by acetone, but not by hydrocarbon solvents. Oxidant-induced reduction of cytochrome b₆ was observed in the lyophilized and hexane-extracted complex, but was lost in the acetone-extracted complex. Reconstitution was achieved either with plastoquinol-1 or plastoquinol-9, suggesting that a plastoquinol molecule is involved in oxidant-induced reduction of cytochrome b₆.     

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b₆f with the electron transfer proteins plastocyanin and cytochrome c₆. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c₆ from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c₆ were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge–charge interactions pre-orient cytochrome c₆ with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.     

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.     

Functional Characterization of the Small Regulatory Subunit PetP from the Cytochrome b6f Complex in Thermosynechococcus elongatus

The cyanobacterial cytochrome b₆f complex is central for the coordination of photosynthetic and respiratory electron transport and also for the balance between linear and cyclic electron transport. The development of a purification strategy for a highly active dimeric b₆f complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled characterization of the structural and functional role of the small subunit PetP in this complex. Moreover, the efficient transformability of this strain allowed the generation of a ΔpetP mutant. Analysis on the whole-cell level by growth curves, photosystem II light saturation curves, and P700⁺ reduction kinetics indicate a strong decrease in the linear electron transport in the mutant strain versus the wild type, while the cyclic electron transport via photosystem I and cytochrome b₆f is largely unaffected. This reduction in linear electron transport is accompanied by a strongly decreased stability and activity of the isolated ΔpetP complex in comparison with the dimeric wild-type complex, which binds two PetP subunits. The distinct behavior of linear and cyclic electron transport may suggest the presence of two distinguishable pools of cytochrome b₆f complexes with different functions that might be correlated with supercomplex formation.     

CYTOCHROME c548 IN NOSTOC SP. (CYANOPHYCEAE): AN ELECTRON ACCEPTOR FROM REDUCED NADP IN THE DARK1

A c-type, carbon monoxide-binding cytochrome was isolated and studied from an endophytic Nostoc sp. Enzymatic transfer of electrons from NADPH to this cytochrome was demonstrated in cell-free preparations under dark, anaerobic conditions, probably mediated by NADPH-cytochrome reductase or ferredoxin-NADP reductase present in the extracts. This cytochrome, with an α-peak at 548 nm, had physicochemical properties similar to the low potential c-type cytochrome, cytochrome c₅₄₉, previously described by other workers from Anacystis nidulans. The participation of such c-cytochromes in dark electron transport, possibly respiratory, of bluegreen algae is strongly indicated.     

Nuclear genes required for post-translational steps in the biogenesis of the chloroplast cytochrome b 6 f complex in maize

Nuclear genes essential for the biogenesis of the chloroplast cytochrome b ₆ f complex were identified by mutations that cause the specific loss of the complex. We describe four transposon-induced maize mutants that lack cytochrome b ₆ f proteins but contain normal levels of other photosynthetic complexes. The four mutations define two nuclear genes. To identify the step at which each mutation blocks protein accumulation, mRNAs encoding each subunit were examined by Northern hybridization analysis and the rates of subunit synthesis were examined in pulse-labeling experiments. In each mutant the mRNAs encoding the known subunits of the complex were normal in size and abundance and the major subunits were synthesized at normal rates. Thus, these mutations block the biogenesis of the cytochrome b ₆ f complex at a post-translational step. The two nuclear genes identified by these mutations may encode previously unknown subunits, be involved in prosthetic group synthesis or attachment, or facilitate assembly of the complex. These mutations were also used to provide evidence for the authenticity of a proposed fifth subunit of the complex and to demonstrate a role for the cytochrome b ₆ f complex in protecting photosystem 11 from light-induced degradation.     

Studies on the cytochrome b6/f complex. I. Characterization of the complex subunits in Chlamydomonas reinhardtii

We have analyzed the heme-associated peroxidase activity in thylakoid membranes from the green algae Chlamydomonas reinhardtii after electrophoresis in the presence of sodium dodecyl sulfate. Besides cytochrome f and cytochrome b₆, we observed peroxidase activity in two other bands, of 34 and 11 kDa, of unknown origin. Characterization of the b₆/f complex subunits was undertaken by means of a comparison of the polypeptide deficiencies in several b₆/f mutants with the polypeptide content of preparations enriched in b₆/f complexes. We conclude that the b₆/f complex consists of five subunits. Using site-specific translation inhibitors, we show that cytochrome f, cytochrome b₆ and subunit IV are of chloroplast origin, whereas the Rieske protein and probably subunit V are translated on cytoplasmic ribosomes. A model of assembly of the complex is proposed: a cytochrome moiety, comprising the subunits of chloroplast origin, is assembled in the thylakoid membranes prior to the insertion and assembly of the subunits encoded in the nuclear genome.     

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1 I. Spectral properties and redox potentials

1. Purified mitochondria have been prepared from wild type Paramecium tetraurelia and from the mutant Cl₁ which lacks cytochrome aa₃. Both mitochondrial preparations are characterized by cyanide insensitivity. Their spectral properties and their redox potentials have been studied.2. Difference spectra (dithionite reduced minus oxidized) of mitochondria from wild type P. tetraurelia at 77 K revealed the α peaks of b-type cytochrome(s) at 553 and 557 nm, of c-type cytochrome at 549 nm and a-type cytochrome at 608 nm. Two α peaks at 549 and 545 nm could be distinguished in the isolated cytochrome c at 77 K. After cytochrome c extraction from wild type mitochondria, a new peak at 551 nm was unmasked, probably belonging to cytochrome c₁. The a-type cytochrome was characterized by a split Soret band with maxima at 441 and 450 nm. The mitochondria of the mutant Cl₁ in exponential phase of growth differed from the wild type mitochondria in that cytochrome aa₃ was absent while twice the quantity of cytochrome b was present. In stationary phase, mitochondria of the mutant were characterized by a new absorption peak at 590 nm.3. Cytochrome aa₃ was present at a concentration of 0.3 nmol/mg protein in wild type mitochondria and ubiquinone at a concentration of 8 nmol/mg protein both in mitochondria of the wild type and the mutant Cl₁. Cytochrome aa₃ was more susceptible to heat than cytochromes b and c,c₁.4. CO difference spectra at 77 K revealed two different Co-cytochrome complexes. The first, found only in wild type mitochondria, was a typical CO-cytochrome a₃ complex characterized by peaks at 596 and 435 nm and troughs at 613 and 450 nm. The second, found both in mitochondria of the wild type and the mutant, was a CO-cytochrome b complex with peaks at 567, 539 and 420 nm and a trough at 558-549 nm. Both complexes are photo-dissociable.5. Spectral evidence was obtained for interaction of cyanide with the a-type cytochrome (shift of the α peak at 77 K from 608 to 605 nm), but not with the b-type cytochrome.6. The mid-point potentials of the different cytochromes at neutral pH are as follows: cytochrome aa₃ 235 and 395 mV, cytochrome c,c₁ 233 mV, cytochromes b 120 mV.     

The Rieske/cytochrome b complex of Heliobacteria

Heliobacteria have a Rieske/cytochrome b complex composed of a Rieske protein, a cytochrome b_6, a subunit IV and a di-heme cytochrome c. The overall structure of the complex seems close to the b₆f complex from cyanobacteria and chloroplasts to the exception of the di-heme cytochrome. We show here by biochemical and biophysical studies that a heme c_i is covalently attached to the Rieske/cytochrome b complex from Heliobacteria. We studied the EPR signature of this heme in two different species, Heliobacterium modesticaldum and Heliobacillus mobilis. In contrast to the case of b₆f complex, a strong axial ligand to the heme is present, most probably a protonatable amino acid residue.     

Cytochrome function in the cyclic electron transport pathway of chloroplasts

Flash excitation of isolated intact chloroplasts promoted absorbance transients corresponding to the electrochromic effect (P-518) and the α-bands of cytochrome b₆ and cytochrome f. Under conditions supporting coupled cyclic electron flow, the oxidation of cytochrome b₆ and the reduction of cytochrome f had relaxation half-times of 15 and 17 ms, respectively. Optimal poising of cyclic electron flow, achieved by addition of 0.1 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea, increased phosphorylation of endogenous ADP and prolonged these relaxation times. The presence of NH₄Cl, or monensin plus NaCl, decreased the half-times for cytochrome relaxation to approximately 2 ms. Uncouplers also revealed the presence of a slow rise component in the electrochromic absorption shift, with formation half-time of about 2 ms. The inhibitors of cyclic phosphorylation antimycin and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone abolished the slow rise in the electrochromic shift and prolonged the uncoupled relaxation times of cytochromes b₆ and f by factors of ten or more.These observations indicate that cytochrome b₆, plastoquinone and cytochrome f participate in a coupled electron transport process responsible for cyclic phosphorylation in intact chloroplasts. Estimations of cyclic phosphorylation rates from 40 to 120 μmol ATP/mg chlorophyll per h suggest that this process can provide a substantial fraction of the ATP needed for CO₂ fixation.     

Characterisation of cytochrome c6from Chlorella fusca

A c-type monohaem, cytochrome c₆was isolated from a soluble extract of the green alga Chlorella fusca. The isolated protein shows an apparent molecular mass of 10 kDa by SDS-PAGE, but behaves as a dimer of 20.3 kDa in gel-filtration; the isoelectric point is 3.6. The N-terminal sequence shows high identity with other green algae cytochromes c₆. The mid-point redox potential is about +350 mV between pH 5 and 9. The ferric and ferrous forms, and their pH equilibria, have been studied using visible, CD and EPR spectroscopies. The visible spectrum of the reduced cytochrome c₆is typical of a c-type haem protein, with maxima at 274 nm, 318 nm (-peak), 416 nm (-peak), 522 nm (-peak), 552–553 nm (-peak). A 690 nm band, characteristic of a haem Met-His axial coordination of the haem group, is present in the oxidized form. At high pH values ( 8), cytochrome c₆undergoes an alkaline transition, with a pKa of 8.7. Between pH 3 and 9 the EPR spectrum is dominated by two rhombic species, with g-values at 3.32, 2.05, 1.05 and 2.96, 2.30, 1.43, which interconvert with a pK_aof 4. CD spectrum of Chlorella fusca cytochrome c₆shows that the proteins must be mainly built up by -helices. Even though there are similarities between Chlorella fusca cytochrome c₆and that isolated from Monoraphidium braunii, no cross-reactivity with the antibodies raised against the Chlorella fusca cytochrome has been detected for the protein from Monoraphidium braunii.     

Characterization of Nuclear Mutants of Maize Which Lack the Cytochrome f/b-563 Complex

Two high fluorescent, nuclear recessive mutants of maize (Zea mays L.), designated hcf-2 and hcf-6, are described which are missing the chloroplast cytochrome f/b-563 complex. Thylakoids from the mutants show a block in whole chain electron transport activity (H₂O to methyl viologen), while retaining activities associated with photosystem II (H₂O to phenylenediamine) and photosystem I (diaminodurene to methyl viologen). Chemically induced, optical difference spectra indicate a loss of cytochromes f and b-563. Cytochrome b-559 is present in both high and low potential forms. EPR analyses of thylakoid membranes of hcf-6 reveals the lack of a signal (g = 1.90) associated with the Rieske Fe-S center. Additionally, hcf-6 is lacking EPR signals at g = 6 (attributable to the high spin ferric heme of cytochrome b-563) and g = 2.5 (unidentified). The mutant retains signals at g = 2.9 (cytochrome b-559) and at g = 4.3 and 9 (both signals probably arising from a storage form of ferric iron).

Thylakoid polypeptides are examined using polyacrylamide gel electrophoresis. hcf-2 and hcf-6 have identical profiles, showing losses of polypeptides with apparent molecular masses of 33 (cytochrome f), 23 (cytochrome b-563), and 17.5 kilodaltons. The protein associated with the Rieske Fe-S center could not be determined from the gel profiles. Additionally, both mutants show an increase in a band with a molecular mass of 31 kilodaltons.

     

Studies on the reaction of imidazole with cytochrome c3 from Desulfovibrio vulgaris

1. Cytochrome c₃, a unique hemoprotein with a negative redox potential and four heme groups bound to a single polypeptide chain, reacts with imidazole in the reduced state to form a low-spin ferro · imidazole complex which is spectrally characterized by a 3.1 nm blue shift in the α-peak (from 550.5 to 547.4 nm). The spectral imidazole · cytochrome c₃ complex is detectable at 77 but not at 298 K.2. Mammalian ferrocytochrome c did not undergo a spectral interaction with imidazole at either 77 or 298 K, indicating that the imidazole · cytochrome c₃ complex reflects a unique event for cytochrome c₃.3. Formation of the imidazole · cytochrome c₃ complex is strongly dependent on imidazole concentration (apparent K_d of approx. 50 mM), and is abolished in the presence of 100 mM phosphate. This latter effect is attributable to formation of an imidazole · phosphate complex. A pH titration of the imidazole · cytochrome c₃ spectral complex implicates ionization of an imidazole function (pK = 8.5).4. EPR studies at 8.5 K of ferricytochrome c₃ before and after one reduction-oxidation cycle indicate that at least two of the hemes undergo reaction with imidazole forming two different low-spin ferric heme · imidazole complexes, with significant shifts in the g values of two heme signals.5. The spectral and EPR results are consistent with formation as the primary event of a low-spin ferrocytochrome c₃ · imidazole complex in which increased hydrophobicity and protonation-deprotonation effects are contributary to the consequent lability of cytochrome c₃.     

Ubiquinol-cytochrome c reductase (EC 1.10.2.2). Isolation in Triton X-100 by hydroxyapatite and gel chromatography. Structural and functional properties

1. A method for the isolation of a monodisperse ubiquinol-cytochrome c reductase (complex III) from beef heart mitochondria has been developed. The procedure consists of an enzyme solubilization in Triton X-100 followed by hydroxyapatite and gel chromatography.2. The minimum unit of the isolated complex is composed of 9 polypeptide subunits with M_r of 49000, 47000, 30000, 25000, 12000, 11000 and 6000. It contains 8 μmol of cytochrome b, 4 μmol of cytochrome c₁ 7–8 μmol of nonheme iron, corresponding to 3.5–4 μmol of the Rieske iron-sulfur protein, less than 1.0 μmol of ubiquinone and about 60 μmol of phospholipids, per g of protein. The specific detergent binding amounts to 0.2 g of Triton X-100 per g protein.3. Cytochrome b exhibits an α-absorbance maximum at 562 nm. In redox titrations it reveals two half-reduction potentials, i.e. ?10 and +100 mV, at pH 7.0. The absorbance maximum of cytochrome c₁ lies at 553 nm and its half-reduction potential amounts to +250 mV.4. The reductase reveals electron-transferring activity with ubiquinol-1, -2, -3, and -9 as donor and cytochrome c as acceptor. The activity with ubiquinol-9 was analyzed according to the surface dilution scheme developed for the action of phospholipases. The molecular activity amounts to 75 mol of cytochrome c reduced per s at 20°C.5. A dissociation constant K′_s of 5.5 mM has been determined for the Triton-solubilized enzyme: ubiquinol-containing micelle association. In this case the total concentration of ubiquinol plus Triton X-100 has been substituted for the concentration of binding areas on the ubiquinol-containing micelles. This substitution makes the reasonable assumption that the sum of ubiquinol concentration plus Triton X-100 is proportional to the number of available binding areas.6. A K′_m value of 0.025 was found for ubiquinol-9. This is an analog to the Michaelis constant and is expressed as mol fraction of ubiquinol in the ubiquinol-Triton micelle.     

The effect of sulphide on cytochromeaa3 Isosteric and allosteric shifts of the reduced α-peak

1. Sulphide, like cyanide, is a slow-binding inhibitor of cytochromeaa₃ with a high affinity (K_d < 0.1 μM).2. Unlike cyanide binding, the binding of sulphide is apparently independent of the redox state of components of the oxidase other than cytochromea₃and shows no anomalous kinetics during complex formation.3. Sulphide binding to cytochrome a₃³⁺ is accompanied by a blue-shift in the α-peak of the reduced enzyme (a²⁺ a₃³⁺H₂S), similar to but smaller than that induced by azide.4. The reduced sulphide-inhibited system shows a much higher Soret peak at 445 nm than the corresponding cyanide and azide complexes, suggesting that partial electron transfer from sulphide to haem may occur in the complex. No evidence was obtained for the formation of any sulfhaem derivatives of cytochromea₃.5. The influence of energization on the spectrum of mitochondrial cytochrome oxidase, and the effects of calcium on the α-peak of isolated cytochromeaa₃ (Wikstro¨m, M. K. F. (1974) Ann. N. Y. Acad. Sci. 227, 146–158) are distinct from the action of the cytochromea₃ligands.6. A classification of peak shifts in the α-region in terms of isosteric and allosteric ligands is proposed.