The methylamine oxidizing system of Pseudomonas AM1 reconstituted with purified components

The electron transport system coupled to the oxidation of methylamine in Pseudomonas AM1 was investigated by reconstituting it from the highly purified components. A mixture of methylamine dehydrogenase, cytochrome cH and cytochrome c oxidase (= cytochrome aa3) actively oxidized methylamine (161 mol of O2 consumed/mol of heme a of cytochrome c oxidase X min). In this system, addition of amicyanin did not affect the oxygen consumption rate. The oxygen consumption rate of the cell-free extract prepared from the cells cultivated in a copper-deficient medium was directly proportional to the amount of amicyanin added, and extrapolation to zero copper concentration gave a value of 28 mol of O2 consumed/mol of heme a of cytochrome c oxidase X min. These results suggest that methylamine oxidation in the bacterium can occur at least to some extent without participation of amicyanin.     

The microbial metabolism of C1 compounds. The electron-transport chain of Pseudomonas AM1

Pseudomonas AM1, Hyphomicrobium X and Pseudomonas MS all contain cytochrome a/a(3) and a b-type cytochrome able to react with CO. Pseudomonas AM1 and Hyphomicrobium X also have a CO-binding cytochrome c. The purified cytochrome c (redox potential 0.26V) of Pseudomonas AM1 was not susceptible to oxidation by molecular oxygen. CO reacted slowly with the reduced form giving a CO difference spectrum with a peak at 412nm and troughs at 420nm and 550nm. Similar results were obtained with the cytochrome c of Hyphomicrobium (aerobically grown or anaerobically grown with nitrate) and with that of Pseudomonas extorquens. The results given in the present paper are incompatible with an oxygenase or oxidase function for the soluble cytochrome c of methylotrophs. Studies with whole cells of Pseudomonas AM1 and a cytochrome c-deficient mutant have demonstrated that cytochrome b (redox potential 0.009V) is the first cytochrome in the electron-transport chain for oxidation of all substrates except methanol (and ethanol) whose oxidation does not involve this cytochrome. All substrates are usually oxidized by way of cytochrome c and cytochrome oxidase (cytochrome a/a(3)), but there is an alternative route for the reduction of cytochrome a/a(3) in the mutant lacking cytochrome c. Results of experiments on cyanide inhibition of respiration and cytochrome oxidation support the suggestion that the susceptibility of cytochrome b to oxidation by molecular oxygen (reflected in its ability to react with CO) is probably irrelevant to the normal physiology of Pseudomonas AM1.     

The respiratory chain of Corynebacterium glutamicum

Corynebacterium glutamicum is an aerobic bacterium that requires oxygen as exogenous electron acceptor for respiration. Recent molecular and biochemical analyses together with information obtained from the genome sequence showed that C. glutamicum possesses a branched electron transport chain to oxygen with some remarkable features. Reducing equivalents obtained by the oxidation of various substrates are transferred to menaquinone via at least eight different dehydrogenases, i.e. NADH dehydrogenase, succinate dehydrogenase, malate:quinone oxidoreductase, pyruvate:quinone oxidoreductase, D-lactate dehydrogenase, L-lactate dehydrogenase, glycerol-3-phosphate dehydrogenase and L-proline dehydrogenase. All these enzymes contain a flavin cofactor and, except succinate dehydrogenase, are single subunit peripheral membrane proteins located inside the cell. From menaquinol, the electrons are passed either via the cytochrome bc(1) complex to the aa(3)-type cytochrome c oxidase with low oxygen affinity, or to the cytochrome bd-type menaquinol oxidase with high oxygen affinity. The former branch is exceptional, in that it does not involve a separate cytochrome c for electron transfer from cytochrome c(1) to the Cu(A) center in subunit II of cytochrome aa(3). Rather, cytochrome c(1) contains two covalently bound heme groups, one of which presumably takes over the function of a separate cytochrome c. The bc(1) complex and cytochrome aa(3) oxidase form a supercomplex in C. glutamicum. The phenotype of defined mutants revealed that the bc(1)-aa(3) branch, but not the bd branch, is of major importance for aerobic growth in minimal medium. Changes of the efficiency of oxidative phosphorylation caused by qualitative changes of the respiratory chain or by a defective F(1)F(0)-ATP synthase were found to have strong effects on metabolism and amino acid production. Therefore, the system of oxidative phosphorylation represents an attractive target for improving amino acid productivity of C. glutamicum by metabolic engineering.     

Purification and properties of the methane mono-oxygenase enzyme system from Methylosinus trichosporium OB3b.

1. A three-component enzyme system that catalyses the oxidation of methane to methanol has been highly purified from Methylosinus trichosporium. 2. The components are (i) a soluble CO-binding cytochrome c, (ii) a copper-containing protein and (iii) a small protein; the mol. wts. are 13 000, 47 000 and 9400 respectively. The cytochrome component cannot be replaced by similar cytochrome purified from Pseudomonas extorquens or by horse heart cytochrome c. 3. The stoicheiometry suggests a mono-oxygenase mechanism and the specific activity with methane as substrate is 6 micronmol/min per mg of protein. 4. Other substrates rapidly oxidized are ethane, n-propane, n-butane and CO. Dimethyl ether is not a substrate. 5. The purified enzyme system utilizes ascorbate or, in the presence of partially purified M. trichosporium methanol dehydrogenase, methanol as electron donor but not NADH or NADPH. 6. Activity is highly sensitive to low concentrations of a variety of chelating agents, cyanide, 2-mercaptoethanol and dithiothreitol. 7. Activity is highly pH-dependent (optimum 6.9-7.0) and no component of the enzyme is stable to freezing. 8. The soluble CO-binding cytochrome c shows oxidase acitivity and the relationship between this and the oxygenase activity is discussed.     

The microbial metabolism of C1 compounds. The stoicheiometry of respiration-driven proton translocation in Pseudomonas AM1 and in a mutant lacking cytochrome c

This paper clarifies the role of cytochrome c in Pseudomonas AM1 by measuring the stoicheiometry of proton translocation driven by respiration of endogenous or added substrates in wild-type bacteria and in a mutant lacking cytochrome c (mutant PCT76). The maximum -->H(+)/O ratio (protons translocated out of the bacteria per atom of oxygen consumed during respiration) was about 4 and, except when respiration was markedly affected, this ratio was similar in mutant and wild-type bacteria. The -->H(+)/O ratios were unaltered when the usual oxidase (cytochrome a(3)) was inhibited by 300mum-KCN and respiration involved the single cytochrome b functioning as an alternative oxidase. Ratios measured in cells respiring endogenous substrate and in cells loaded with malate or 3-hydroxybutyrate suggest that there are two proton-translocating segments operating during the oxidation of NADH. By contrast, during oxidation of formaldehyde or methylamine only one pair of protons is translocated. Proton translocation could not be measured with methanol as substrate, because its oxidation was inhibited (90-95%) by 5mm-KSCN. It is tentatively proposed that the electron-transport chain for NADH oxidation in Pseudomonas AM1 is arranged such that the NADH-ubiquinone oxidoreductase forms one proton-translocating segment and the second segment consists of ubiquinone and cytochromes b and a/a(3). The cytochrome c appears to be essential only for respiration and proton translocation from methanol (and possibly from methylamine); there is no conclusive evidence that cytochrome c ever mediates between cytochromes b and a/a(3) in Pseudomonas AM1.     

The reaction of the electrostatic cytochrome c-cytochrome oxidase complex with oxygen.

The reaction of the electrostatic cytochrome c-cytochrome oxidase complex with oxygen is measured by transient absorption spectroscopy. The oxygen reaction is initiated by photolytic removal of CO from cytochrome oxidase, using a flash-pumped dye laser. The subsequent reaction of the cytochrome c-cytochrome oxidase complex with oxygen is reported at 550, 605, 744, and 830 nm at different cytochrome c:cytochrome oxidase ratios and different oxygen concentrations. In the absence of cytochrome c the time course of the reaction of the oxidase is well described by a triple exponential process at any of the measured wavelengths. The three processes are well resolved at high O2 levels (i.e. greater than 200 microM), where they reach first-order rate limits of 2.4 x 10(4), 7.5 x 10(3), and 650 s-1. When cytochrome c is added the oxidation of cytochrome a and one of the redox active cooper centers (CuA) are interrupted. The maximal effect of cytochrome c on the oxidation of the oxidase occurs at a c:aa3 ratio of 1. Cytochrome c reacts in a biphasic process with rates of up to 7 x 10(3) and 550 s-1 at high oxygen. The fast phase takes up 60% of the process, and this is independent of the cytochrome c:cytochrome oxidase ratio. The results are discussed in the context of a model in which electron entry into cytochrome oxidase from cytochrome c is via CuA, and cytochrome a functions to mediate electron transfer from CuA to the oxygen binding site. The role of CuA as initial electron acceptor in cytochrome c oxidase is related to its physical proximity to cytochrome c is the cytochrome c-cytochrome oxidase complex.     

The terminal respiratory chain of the methylotrophic bacterium Methylophilus methylotrophus

Cytochrome oxidase o has been isolated from the obligately aerobic, methylotrophic bacterium Methylophilus methylotrophus in the form of a cytochrome cL-o complex. The latter is comprised of cytochrome cL (Mr 21 000) and cytochrome o (Mr 29 000) in a 1-2:1 ratio, possibly in association with one or more minor polypeptides; the complex exhibits a high ascorbate-TMPD oxidase activity which is inhibited non-competitively by cyanide (Ki approximately 2 microM). In contrast, the oxidation of methanol by whole cells is inhibited uncompetitively by cyanide (Ki approximately 4 microM), thus indicating the involvement in methanol oxidation of cytochrome oxidase aa3 rather than o.     

Sulphide as an inhibitor and electron donor for the cytochrome c oxidase system

Anomalies both kinetic and equilibrium in nature are described for the inhibition of cytochrome c oxidase activity by sulphide in the isolated enzyme and in submitochondrial particles. These anomalies are related to the involvement of more than 1 mol of sulphide in the blockage of one cytochrome aa3 centre. Sulphide reduces resting cytochrome a3, a reaction that results in oxygen uptake and the loss of a sulphide molecule. Sulphide can also reduce cytochromes c and a; in the former case, a part of the one-equivalent oxidation product, presumed to be the SH radical, reacts with oxygen. Such oxygen uptake is also seen under aerobic conditions when ferricyanide reacts with sulphide. Three phases are identified in the inhibitory interaction of sulphide with the cytochrome c oxidase enzyme itself: an initial rapid reaction involving sulphide oxidation, oxygen uptake, and conversion of cytochrome aa3 into the low-spin "oxyferri" form; a subsequent step in which sulphide reduces cytochrome a; and the final inhibitory step in which a third molecule of sulphide binds the a3 iron centre in the cytochrome a2+ a3 3+ (oxy) species to give cytochrome a2+ a3 3+ H2S. the initial events parallel some of the events in the interaction of the cytochrome c-cytochrome aa3 system with monothiols; the final inhibitory event resembles that with cyanide.     

Nucleotide sequence of the Methylobacterium extorquens AM1 moxF and moxJ genes involved in methanol oxidation

The nucleotide sequence has been determined for two genes involved in methanol oxidation in the facultative methylotroph, Methylobacterium extorquens AM1. The two genes are moxF, encoding the 66-kDa subunit of the methanol dehydrogenase and moxJ, located immediately downstream from moxF, which encodes a 30-kDa protein with unknown function. This information completes the sequence of the 5.86-kb XhoI-SalI fragment containing the moxFJGI region in M. extorquens AM1, and the structure of this gene cluster is presented. Evidence is presented that moxJ is also present in Paracoccus denitrificans. The aa sequence of MoxJ has provided little information concerning its function, but it does appear to contain a signal sequence suggesting a periplasmic location.     

Characterization of a novel soluble c-type cytochrome in a moxD mutant of Methylobacterium extorquens AM1

Methylobacterium extorquens AM1 contains a novel c-type cytochrome, called cytochrome c-553, previously thought to be a precursor of the electron acceptor (cytochrome cL) for methanol dehydrogenase. Its amino acid composition and serological characteristics show that it has no structural relationship to cytochrome cL. It usually comprises less than 5% of the total c-type cytochromes. In a moxD mutant, which contains neither methanol dehydrogenase nor cytochrome cL, it comprises 30% of the soluble cytochrome and it has been purified and characterized from that mutant. Cytochrome c-553 is large (Mr 23,000), acidic and monohaem, with a redox potential of 194 mV. It reacts rapidly and completely with CO but is not autoxidizable. It is not autoreducible, and it is not an electron acceptor from methanol dehydrogenase or methylamine dehydrogenase, nor an important electron donor to the oxidase. It is able to accept electrons from cytochrome cL and to donate electrons to cytochrome cH. It is present in the soluble fraction (presumably periplasmic) and membrane fraction of wild-type bacteria during growth on a wide range of growth substrates, but its function in these bacteria or in the moxD mutant has not been determined.     

One of two copper atoms is not necessary for the cytochrome c oxidase activity of Pseudomonas AM 1 cytochrome aa3

From Pseudomonas AM 1 grown in a medium deficient in Cu, aa3-type cytochrome c oxidase was purified which contained 2 molecules of haem a and one atom of Cu per molecule. The enzyme showed absorption peaks at 428 and 595 nm in the oxidized form and at 442 and 604 nm in the reduced form, and its CO complex showed peaks at 432 and 602 nm. The enzyme in the oxidized state showed an obscure absorption peak around 800 nm instead of a peak at 820 nm. One mol of the enzyme oxidized maximally 76, 75, and 98 mol of the ferrocytochromes c of Candida krusei, horse and Pseudomonas AM 1 per sec, respectively. These reactions were 50% inhibited by 7 microM KCN. The product of reduction of O2 catalyzed by the enzyme was concluded to be H2O on the basis of the ratio of ferrocytochrome c oxidized to O2 consumed.     

Kinetics of the interaction of the cytochrome c oxidase of Paracoccus denitrificans with its own and bovine cytochrome c

We have devised a relatively simple method for the purification of cytochrome aa3 of Paracoccus denitrificans with three major subunits similar to those of the larger subunits of the mitochondrial cytochrome oxidase. This preparation has no c-type cytochrome. Studies were made of the oxidation of soluble cytochromes c from bovine heart and Paracoccus. The cytochrome-c oxidase activity was stimulated by low concentrations of either cytochrome c, providing an explanation for the multiphasic nature of plots of v/S versus v. Kinetics of the oxidation of bovine cytochrome c by the Paracoccus oxidase resembled those of bovine oxidase with bovine cytochrome c in every way; the Paracoccus oxidase with bovine cytochrome c can serve as an appropriate model for the mitochondrial system. The kinetics of the oxidation of the soluble Paracoccus cytochrome c by the Paracoccus oxidase were different from those seen with bovine cytochrome c, but resembled the latter if poly(L-lysine) was added to the assays. The important difference between the two species of cytochrome c is the more highly negative hemisphere on the side of the molecule way from the heme crevice in the Paracoccus cytochrome. Thus, the data emphasize the importance of all of the charged groups on cytochrome c in influencing the binding or electron transfer reactions of this oxidation-reduction system. The data also permit some interesting connotations about the possible evolution from the bacterial to the mitochondrial electron transport system.     

Isolation and characterization of the moxJ, moxG, moxI, and moxR genes of Paracoccus denitrificans: inactivation of moxJ, moxG, and moxR and the resultant effect on methylotrophic growth.

By using the moxF gene encoding the large fragment of methanol dehydrogenase as a probe, a downstream linked chromosomal fragment was isolated from a genomic bank of Paracoccus denitrificans. The nucleotide sequence of the fragment was determined and revealed the 3' part of moxF, four additional open reading frames, and the 5' part of a sixth one. The organization and deduced amino acid sequences of the first three frames downstream from moxF were found to be largely homologous to the moxJ, moxG, and moxI gene products of Methylobacterium extorquens AM1. Directly downstream from these three genes, a new mox gene was identified. The gene is designated moxR. By using the suicide vector pGRPd1, the moxJ, moxG, and moxR genes were inactivated by the insertion of a kanamycin resistance gene. Subsequently, suicide vector pRVS1 was used to replace the marker genes in moxJ and moxG for unmarked deletions made in vitro. As a result, the three insertion strains as well as the two unmarked mutant strains were unable to grow on methanol, even in the presence of pyrroloquinoline quinone. Growth on succinate and on methylamine was not affected. In all five mutant strains, synthesis of the large subunit of methanol dehydrogenase and of inducible cytochrome c553i was observed. The moxJ and moxG insertion mutant strains were unable to synthesize both the cytochrome c551i and the small subunit of methanol dehydrogenase, and this lack of synthesis was attended by the loss of methanol dehydrogenase activity. The moxJ deletion mutant strain partly synthesized the latter two proteins, cytochrome c551i. Partial synthesis of the small subunit of methanol dehydrogenase observed with the latter strain was attended by a corresponding extent of methanol dehydrogenase activity. The moxR insertion mutant strain was shown to synthesize cytochrome c551i as well as the large and small subunits of methanol dehydrogenase, but no methanol dehydrogenase activity was observed. The results show that periplasmic cytochrome c551i is the moxG gene product and the natural electron acceptor of methanol dehydrogenase in P. denitrificans. In contrast to earlier suggestions, this cytochrome was found to be different from membrane-bound cytochrome c552. In addition, it is demonstrated that moxI encodes the small subunit of methanol dehydrogenase. It is suggested that MoxJ is involved in the assemblage of active methanol dehydrogenase in the periplasm and, in addition, that MoxR is involved in the regulation of formation of active methanol dehydrogenase.     

Electron transport reactions in a cytochrome c-deficient mutant of Paracoccus denitrificans

A mutant of Paracoccus denitrificans which is deficient in c-type cytochromes grows aerobically with generation times similar to those obtained with a wild-type strain. The aa3-type oxidase is functional in the mutant as judged by spectrophotometric assays of cytochrome c oxidation using the membrane particles and cytochrome aa3 reduction in whole cells. The cytochrome c oxidase (aa3-type) of the c-less mutant oxidizes soluble cytochrome c at rates equivalent to those obtained with the wild-type. NADH and succinate oxidase activities of the membrane preparations of the mutant and wild-type are also comparable in the absence of detergent treatment. Exogenous soluble cytochrome c can be both reduced by NADH- and succinate-linked systems and oxidized by cytochrome aa3 present in membranes of the mutant strain. Rapid overall electron transport can occur in the c-less mutant, suggesting that reactions result from collision of diffusing complexes.     

Terminal oxidases in the trypanosomatid Trypanosoma cruzi

Titration of Trypanosoma cruzi respiration with cyanide, with results treated as Dixon plots, indicated the presence of several terminal oxidases. The inhibitions obtained at low cyanide concentrations (0-300 microM), taken together with cyanide effects on cytochrome aa3-deficient, dyskinetoplastic epimastigotes, supported cytochrome aa3 as T. cruzi main terminal oxidase. By increasing cyanide concentration to 1.0 mM, two alternative terminal oxidases could be detected. One of these was active in both kinetoplastic and dyskinetoplastic (cytochrome aa3-deficient) epimastigotes, and azide- and antimycin-insensitive. Complementary cytochrome studies with intact epimastigotes and mitochondrial membranes revealed the presence of cytochromes aa3, b, c558, o and possibly d, as components of the parasite electron transport system. Fractionation studies demonstrated that both o and d were bound to the mitochondrial membrane. Reduction by endogenous substrates and complex formation with cyanide supported cytochrome o as alternative terminal oxidase. EB-cultured, dyskinetoplastic epimastigotes showed the same respiration rate as the kinetoplastic cells, despite the significant decrease of cytochrome aa3, thus indicating adaptive mechanisms that determine the expression of alternative oxidases, whenever the main terminal activity is depressed.     

Transient-state reduction and steady-state kinetic studies of menaquinol oxidase from Bacillus subtilis, cytochrome aa3-600 nm. Spectroscopic characterization of the steady-state species.

Cytochrome aa3-600 or menaquinol oxidase, from Bacillus subtilis, is a member of the heme-copper oxidase family. Cytochrome aa3-600 contains cytochrome a, cytochrome a3, and CuB, and each is coordinated via histidine residues to subunit I. Subunit II of cytochrome aa3-600 lacks CuA, which is a common feature of the cytochrome c oxidase family members. Anaerobic reduction of cytochrome aa3-600 by the substrate analogue 2,3-dimethyl-1,4-naphthoquinone (DMN) resolves two distinct kinetic phases by stopped-flow, single-wavelength spectrometry. Global analysis of time-resolved, multiwavelength spectra shows that during these distinct phases cytochromes a and a3 are both reduced. Cyanide binding to cytochrome a3 enhances the fast phase rate, which in the presence of cyanide can be assigned to cytochrome a reduction, whereas cytochrome a3-cyanide reduction is slow. The steady-state activity of cytochrome aa3-600 exhibits saturation kinetics as a function of DMN concentration with a Km of 300 microM and a maximal turnover of 63.5 s(-1). Global kinetic analysis of steady-state spectra reveals a species that is characteristic of a partially reduced oxygen adduct of cytochrome a3-CuB, whereas cytochrome a remains oxidized. Electron paramagnetic resonance (EPR) spectroscopy of the oxidase in the steady state shows the expected signal from ferricytochrome a, and a new EPR signal at g = 2.01. A model of the catalytic cycle for cytochrome aa3-600 proposes initial electron delivery from DMN to cytochrome a, followed by rapid heme to heme electron transfer, and suggests possible origins of the radical signal in the steady-state form of the enzyme.     

A comparative survey of several bacterial aa3-type cytochrome c oxidases

The aa3-type cytochrome c oxidases purified from Nitrobacter agilis, Thiobacillus novellus, Nitrosomonas europaea, and Pseudomonas AM 1 were compared. They have haem a and copper atom as the prosthertic groups and show alpha and gamma absorption peaks at around 600 and 440 nm, respectively. Each oxidase molecule is composed of two kinds of subunits. The N. agilis oxidase has 2 moles of haem a and 2 atoms of copper in the minimal structural unit composed of one molecule each of the two kinds of subunits, while the T. novellus enzyme seems to contain one molecule of the haem and one atom of the metal in the unit. The N. europaea oxidase shows very low affinity for carbon monoxide. Each oxidase reacts rapidly with some eukaryotic cytochromes c as well as with its native cytochrome c. The cytochrome c oxidase activity of the N. agilis oxidase is 50% inhibited by 1 microM KCN, while 50% inhibition of the activity requires 100 microM KCN in the case of the N. europaea enzyme.     

The nitrite oxidizing system of Nitrobacter winogradskyi

Cytochrome components which participate in the oxidation of nitrite in Nitrobacter winogradskyi have been highly purified and their properties studied in detail. Cytochrome a1c1 is an iron-sulphur molybdoenzyme which has haems a and c and acts as a nitrite-cytochrome c oxidoreductase. Cytochrome c-550 is homologous to eukaryotic cytochrome c and acts as the electron mediator between cytochrome a1c1 and aa3-type cytochrome c oxidase. The oxidase is composed of two kinds of subunits, has two molecules of haem a and two atoms of copper in the molecule, and oxidizes actively eukaryotic ferrocytochrome c as well as its own ferrocytochrome c-550. Further, a flavoenzyme has been obtained which has transhydrogenase activity and catalyses reduction of NADP+ with benzylviologen radical. This enzyme may be responsible for production of NADPH in N. winogradskyi. The electron transfer against redox potential from NO2- to cytochrome c could be pushed through prompt removal by cytochrome aa3 of H+ formed by the dehydrogenation of NO2- + H2O. As cytochrome c in anaerobically kept cell-free extracts is rapidly reduced on addition of NO2-, a membrane potential does not seem necessary for the reduction of cytochrome c by cytochrome a1c1 with NO2- in vivo.     

Pathways of cytochrome c oxidation by soluble and membrane-bound cytochrome aa3

In media of low ionic strength, membraneous cytochrome c oxidase, isolated cytochrome c oxidase, and proteoliposomal cytochrome c oxidase each bind cytochrome c at two sites, one of low affinity (1 microM greater than Kd' greater than 0.2 microM) and readily reversible and the other of high affinity (0.01 microM greater than Kd) and weakly reversible. When cytochrome c occupies both sites, including the low affinity site, the maximal turnover measured polarographically with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) is independent of TMPD concentration, and lies between 250 and 400 s-1 (30 degrees C, pH 7.4) for fully activated systems. The apparent affinity of the enzyme for cytochrome c is, however, TMPD dependent. When cytochrome c occupies only the high-affinity site, the maximal turnover is closely dependent upon the concentration of TMPD, which, unlike ascorbate, can reduce bound cytochrome c. As TMPD concentration is increased, the maximal turnover approaches that seen when both sites as occupied. The lower activity of isolated cytochrome aa3 is due to the presence of inactive or inaccessible enzyme molecules. Incorporation of isolated enzyme into phospholipid vesicles restores full activity to all the subsequently accessible cytochrome aa3 molecules. Negatively charged (asolectin) vesicles show a higher cytochrome c affinity at the low-affinity sites than do the other enzyme preparations. A model for the cytochrome c-cytochrome aa3 complexes is put forward in which both sites, when occupied, are fully catalytically competent, but in which occupation of the "tight" site by a catalytically functional cytochrome c molecule is required for overall oxidation of cytochrome c via the "loose" site.