Bimetallic Center Research Articles

Thermal stability of mixed-valent diruthenium (II,III) carboxylates

The thermal stability of mixed-valent diruthenium carboxylates, Ru 2 II,III (O 2CR) 4X, where RCO 2 − are the equatorial carboxylates and X − is the axial ligand (halide or carboxylate anion) was investigated. The nature of the axial ligands determines two decomposition pathways, while the equatorial carboxylate plays a minor role. The general behavior involves the reduction of the bimetallic center to Ru 0. The halocomplexes (X −=halide) decompose in just one complex step. The pentacarboxylates (X −=CH 3(CH 2) n−2 CO 2 −) have a two-step mechanism being the intermediate product Ru 2 II,II (O 2C(CH 2) n−2 CH 3) 4. The influence of the liquid crystalline phase was explored.

Alkaline phosphatase revisited: hydrolysis of alkyl phosphates.

Escherichia coli alkaline phosphatase (AP) is the prototypical two metal ion catalyst with two divalent zinc ions bound approximately 4 A apart in the active site. Studies spanning half a century have elucidated many structural and mechanistic features of this enzyme, rendering it an attractive model for investigating the potent catalytic power of bimetallic centers. Unfortunately, fundamental mechanistic features have been obscured by limitations with the standard assays. These assays generate concentrations of inorganic phosphate (P(i)) in excess of its inhibition constant (K(i) approximately 1 muM). This tight binding by P(i) has affected the majority of published kinetic constants. Furthermore, binding limits k(cat)/K(m) for reaction of p-nitrophenyl phosphate, the most commonly employed substrate. We describe a sensitive (32)P-based assay for hydrolysis of alkyl phosphates that avoids the complication of product inhibition. We have revisited basic mechanistic features of AP with these alkyl phosphate substrates. The results suggest that the chemical step for phosphorylation of the enzyme limits k(cat)/K(m). The pH-rate profile and additional results suggest that the serine nucleophile is active in its anionic form and has a pK(a) of < or = 5.5 in the free enzyme. An inactivating pK(a) of 8.0 is observed for binding of both substrates and inhibitors, and we suggest that this corresponds to ionization of a zinc-coordinated water molecule. Counter to previous suggestions, inorganic phosphate dianion appears to bind to the highly charged AP active site at least as strongly as the trianion. The dependence of k(cat)/K(m) on the pK(a) of the leaving group follows a Brønsted correlation with a slope of beta(lg) = -0.85 +/- 0.1, differing substantially from the previously reported value of -0.2 obtained from data with a less sensitive assay. This steep leaving group dependence is consistent with a largely dissociative transition state for AP-catalyzed hydrolysis of phosphate monoesters. The new (32)P-based assay employed herein will facilitate continued dissection of the AP reaction by providing a means to readily follow the chemical step for phosphorylation of the enzyme.

Tetranuclear Complexes Containing Bimetallic Tetracarboxylates and Ferrocene − Models for Subunits of One-Dimensional Organometallic Polymers

The tetranuclear compounds M2(O2C-R)4L2 [M = Rh, R = nC7H15, L = ferrocenyl-4-pyridylacetylene (FPA) (1a); M = Rh, R = CH3, L = FPA (1b); M = Mo, R = CF3, L = FPA (2b); M = Rh, R = nC7H15, L = 4-ferrocenylpyridine (FP) (3a); M = Rh, R = CH3, L = FP (3b); M = Mo, R = nC7H15, L = FP (4a); M = Mo, R = CF3, L = FP (4b)] have been synthesized by the reaction of bimetallic tetracarboxylates with L in a 1:2 ratio, in a noncoordinating solvent. The reaction of Mo2(O2C-nC7H15)4 with FPA in a 1:2 molar ratio leads to a trinuclear compound Mo2(O2C-nC7H15)4(FPA) (2a). A soluble oligomer [Rh2(O2C-nC7H15)4(BPEF)]n [5, BPEF = 1,1′-bis(4-pyridylethynyl)ferrocene] is obtained by the reaction of Rh2(O2CnC7H15)4 with BPEF in a 1:1 ratio in CH2Cl2 or CHCl3, whereas the reaction of Mo2(O2C-nC7H15)4 with BPEF leads to a pentanuclear compound [Mo2(O2C-nC7H15)4]2(BPEF) (6), even when BPEF is in excess. 1H NMR, Raman, UV/Vis absorption spectroscopy and cyclic voltammetry indicate that the metal-metal bonds are weakened by the metal-axial ligand interaction and the donor-acceptor interactions occurring between the building blocks of the multinuclear compounds and oligomers. The interaction between the bimetallic centers and the ferrocene moiety is enhanced by shortening the distances between them. This shortening is achieved by modification of the axial ligand from FPA to FP. A significant electronic communication between the ferrocene units is not observed.

Study on the mechanism of anionic polymerization with mixed RLi-R′OK initiators, 2. Theoretical study of random styrene-butadiene copolymerization

Styrene-butadiene copolymerization using mixed RLi-R′OK initiators was modelled on the basis of a traditional two-state scheme which supposes separate polymerization via lithium and potassium active species accompanied by a fast exchange of their counter-ions. Copolymer composition and the monomer-copolymer composition curves were calculated with respect to the influence of the K/Li ratio. Furthermore, the influence of the initial RLi concentration on the K/Li ratio for an azeotropic copolymerization was investigated. None of the proposed relations were found to agree with the published experimental data. Therefore, similar to butadiene homopolymerization, styrene-butadiene copolymerization using an alkyllithium – potassium alkoxide initiator can be rather explained by an one-state polymerization process under the participation of complex bimetallic centres than by a two-state process via individual organometallic compounds.

"Bridging hydroxide effect" on mu-carboxylato coordination and electrochemical potentials of bimetallic centers: Mn2(II,II) and Mn2(III,III) complexes as functional models of dimanganese catalases.

Synthesis, solution structures, and electrochemistry of several dinuclear Mn2(II,II) complexes (1-4) and Mn2(III,III) complexes (6 and 8), derived from a functional catalase mimic [(L1,2)Mn2(II,II)(mu 13-O2CCH3)]2+ (1) are described that enable testing of the role of intramolecular hydroxide ligands on the redox properties. Addition of 1 equiv of hydroxide to 1 or 3 forms [(L1,2)Mn2(II,II)(mu 13-O2CCH3)(mu- OH)]+ (7A or 7B, respectively), possessing two six-coordinate Mn(II) ions bridged by hydroxide and acetato ligands. Two-electron oxidation of 7 with O2 occurs by forming [(L1,2)Mn2(III,III)(mu 1,3-O2CCH3)(mu-OH)]3+ (8) and H2O2 with no ligand rearrangements in methanol. Reaction of 8 with 2-3 equiv hydroxide forms [(L1,2)Mn2(III,III)(mu-O)(OH)(O2CCH3)]+ in which deprotonation of mu-OH- to yield mu-O2- favors subsequent addition of a terminal hydroxide ligand, accommodated by a bridging-to-terminal "carboxylate-shift". Preservation of six-coordinate Mn(II) ions throughout all hydroxide-induced transformations is observed, including oxidation by O2. Cyclic voltammetry reveals that addition of mu-OH- converts the two-electron redox couple II,II/III,III for complexes 1-4 to sequential one-electron couples at lower reduction potentials, yielding substantial stabilization of the II,III and III,III oxidation states by delta E = 440 and 730 mV, respectively. Binding of a second OH- to 7A or 7B forms (L1,2)Mn2(II,II)(mu 13-O2CCH3)(OH)2, containing two six-coordinate Mn(II) ions with two terminal hydroxides and a mu 1,3-bridging acetato. Electrochemistry reveals that displacement of the bridging hydroxide to a terminal site upon addition of the second OH- restores a two-electron redox couple II,II/III,III but now at a higher reduction potential with considerable loss of the electrochemical stabilization energy provided by the mu-OH- (delta E = 250 and 350 mV loss for Mn2(II,II) and Mn2(III,III), (respectively). These results indicate a considerably stronger influence of bridging vs terminal hydroxide ligands in stabilizing the higher oxidation states and separating the one-electron redox potentials of bimetallic centers. By contrast, in the absence of mu-OH- bridges the longer separation with the mu 1,3-carboxylato bridge in dimanganese(II,II) complexes leads to nearly complete uncoupling of the Mn(II) oxidation potentials, thus yielding a two-electron redox transition to (III,III). We hypothesize that this "bridging hydroxide effect" may be due to both greater screening of the repulsive intermetallic electric potential energy and increased resonance stabilization of the mixed-valence (II,III) oxidation state by charge delocalization . These data provide a physicochemical basis for interpretation of the catalase activity of these complexes and of dimanganese catalase enzymes (see the following manuscript).

EQUILIBRIUM AND HYDROLYSIS STUDIES OF PHOSPHATE ESTERS MODEL MOLECULES AND DNA CATALYZED BY OBISDIEN-Zn(II) COMPLEXES

Dinuclear Zn(II)-OBISDIEN complexes catalyze hydrolysis of DNA and a phosphate ester model molecule: bis(2,4-dinitrophenyl)phosphate (BDNPP). The increase in the rate of hydrolysis of BDNPP at pH values above 8.0 is attributed to the presence of a ternary hydroxide species, in which intramolecular catalysis is favored by the proximity of the hydroxide group coordinated to the bimetallic center of the receptor complexes, in agreement with equilibrium results of OBISDIEN-Zn(II)-ATP (1:2:1 molar ratio) system. In the treatment of pBR322 circular DNA with OBISDIEN-Zn(II) complexes, the results suggest that these complexes cleave DNA in a random and nonspecific manner since no distinguishable low molecular weight bands were observed after treatment

Electronic, Magnetic, and Spectroscopic Properties of Binuclear Diruthenium Tetracarboxylates: A Theoretical and Experimental Study

The electronic structure of binuclear diruthenium tetracarboxylates, in both the divalent Ru2II,II(O2CR)4 and the mixed-valent Ru2II,III(O2CR)4X (X = anion) states is studied by means of ZINDO/S-MRCI and DFT calculations. Both methods predict a (π*)2(δ*)2 ground-state configuration for the divalent species, contrary to the (π*)3(δ*)1 previously predicted by SCF-Xα calculations, but in agreement with magnetic measurements that show a strong Zero Field Splitting. Our ZINDO/S-MRCI calculations on compounds containing axial ligands with different degree of π bonding to the bimetallic center (water, chloride, carboxylate, pyridine, pyrazine), for both (II,II) and (II,III) ruthenium cores, show the important role played by the ligands. These theoretical calculations allow us to explain the differences observed in the UV/vis and resonance Raman spectra, both in solution and in the solid state, when varying the axial ligands. The ZINDO/S-MRCI calculations are also capable of solving some controversies found in th...

A Hybrid Density Functional Theory/Molecular Mechanics Study of Nickel−Iron Hydrogenase: Investigation of the Active Site Redox States

We have investigated using theoretical methods some of the redox states of the active site of Desulfovibrio gigas NiFe hydrogenase, which is a metalloprotein that catalyzes the reversible reaction H2 ⇌ 2H+ + 2 e-. A hybrid potential that combines ab initio density functional theory and a molecular mechanics energy function was employed. Starting from the X-ray structure of the oxidized form refined at 2.54 Å resolution, we have optimized the structures of the active site redox states, believed to be involved in the activation and the catalytic cycle of the enzyme, and compared them with the available X-ray data. We have also tested various hypotheses concerning the oxidation states of the Ni−Fe bimetallic center and the protonation states of the active site by comparing calculated spin densities and vibrational frequencies with EPR and IR spectroscopic data. The good agreement we have obtained with experiment allows us to identify more precisely those structures that are likely to be important in the enzymatic reaction mechanism.

The active site of the bacterial nitric oxide reductase is a dinuclear iron center.

A novel, improved method for purification of nitric oxide reductase (NOR) from membranes of Paracoccus denitrificans has been developed. The purified enzyme is a cytochrome bc complex which, according to protein chemical and hydrodynamic data, contains two subunits in a 1:1 stoichiometry. The purified NorBC complex binds 0.87 g of dodecyl maltoside/g of protein and forms a dimer in solution. Similarly, it is dimeric in two-dimensional crystals. Images of these crystals have been processed at 8 A resolution in projection to the membrane. The NorB subunit is homologous to the main catalytic subunit of cytochrome oxidase and is predicted to contain the active bimetallic center in which two NO molecules are turned over to N2O. Metal analysis and heme composition implies that it binds two B-type hemes and a nonheme iron but no copper. NorC is a membrane-anchored cytochrome c. Fourier transform infrared spectroscopy shows that carbon monoxide dissociates from the reduced heme in light and associates with another metal center which is distinct from the copper site of heme/copper oxidases. Electron paramagnetic resonance spectroscopy reveals that NO binds to the reduced enzyme under turnover conditions giving rise to signals near g = 2 and g = 4. The former represents a typical nitrosyl-ferroheme signal whereas the latter is a fingerprint of a nonheme iron/NO adduct. We conclude that the active site of NOR is a dinuclear iron center.

η1−η2 Rearrangement and Protonation of Phenyldiazo Bridging Ligands Attached to the Dimolybdenum System {Mo2Cp2(μ-SMe)3}

Rearrangements and protonation reactions of phenyldiazo ligands bound to the bimetallic center, {Mo2Cp2(μ-SMe)3}, are reported.

Substitution of lysine-362 in a putative proton-conducting channel in the cytochrome c oxidase from Rhodobacter sphaeroides blocks turnover with O2 but not with H2O2.

The recently reported X-ray structures of cytochrome oxidase reveal structures that are likely proton-conducting channels. One of these channels, leading from the negative aqueous surface to the heme a3/CuB bimetallic center, contains a lysine as a central element. Previous work has shown that this lysine (K362 in the oxidase from Rhodobacter sphaeroides) is essential for cytochrome c oxidase activity. The data presented demonstrate that the K362M mutant is impeded in the reduction of the heme a3/CuB bimetallic center, probably by interfering with the intramolecular movement of protons. The reduction of the heme-copper center is required prior to the reaction with dioxygen to form the so-called peroxy intermediate (compound P). This block can be by-passed to some extent by the addition of H2O2, which can react with the enzyme without prereduction of the heme-copper center and can then be reduced to water using electrons from cytochrome c. Hence, the K362M mutant, though lacking oxidase activity, exhibits cytochrome c peroxidase activity. Rapid mixing techniques have been used to determine the kinetics of this peroxidase activity at concentrations of H2O2 up to 0.5 M. The Km for peroxide is about 50 mM and the Vmax is 50 electrons s-1, which is considerably slower than the turnover that can be obtained for the oxidase activity of the wild-type enzyme (1200 s-1). The turnover of the mutant oxidase with H2O2 appears to be limited by the rate of reaction of the enzyme with peroxide to form compound P, rather than the rate of reduction of compound P to water by cytochrome c. The data require a reexamination of the proposed roles of the putative proton-conducting channels.

Smectic bimetallomesogens in β-enaminoketonate copper complexes: the steric effect of a methyl substituent on the core group and the crystal structure

A systematic study has been made of the liquid crystalline properties of two types of copper complexes derived from enamino-β-ketonate and iminopropyl-β-ketonate Schiff bases that exhibit mesomorphism. These complexes are structurally similar, and all have a bimetallic center as an expanded core group. The mesophase stability depends upon the steric bulk between the layer structures. The enamino-β-ketonate complexes exhibit smectic A phases, however, the iminopropyl-β-diketonates are not liquid crystalline. Examination of the structural features of these complexes revealed that the presence of a methyl group attached to the core group was the cause for the lack of liquid crystallinity observed in the latter. The crystal and molecular structure of bis[3,5-dioctanoylphenyl 2-(3-hydroxypropyl)iminopropyl ketonato]copper(II) was determined by means of X-ray analysis. The Cu· · ·Cu intramolecular separation is 3.0375(5) A. The planar geometry is slightly distorted toward square pyramidal by displacement of the copper by 0.018 A from the mean plane through atoms N(1), O(2), O(1A) and O(1).

Synthesis and Reactivities of Cationic Diruthenium Complexes with Terminal Vinylidene Ligands. Hydration and Novel Cyclization of Acetylenes on the Diruthenium Center

The cationic diruthenium complexes with a terminal vinylidene ligand [Cp*RuCl(μ2-SPri)2Ru(CCHR)Cp*][OTf] (3a, R = COOMe; 3b, R = COMe; 3c, R = H; Cp* = η5-C5Me5, OTf = OSO2CF3) were obtained in high yields by the reaction of [Cp*RuCl(μ2-SPri)2Ru(OH2)Cp*][OTf] with HC⋮CR. Hydration of 3a and 3b gave the carbonyl complex [Cp*RuCl(μ2-SPri)2Ru(CO)Cp*][OTf] and CH3R in almost quantitative yields, while the reaction of 3c with water gave the μ2-acetyl complex [Cp*RuCl(μ2-SPri)2{μ2-C(O)Me}RuCp*][OTf] (5). A reaction mechanism for the former reactions is proposed, which includes hydration of the vinylidene ligands to form the μ2-β-ketoacetyl complexes with a six-membered dimetallacyclic structure [Cp*Ru(μ2-SPri)2(η1:η1-μ2-COCH2COR‘)RuCp*]+ (R‘ = OMe, Me) and the following decarbonylation reaction of the μ2-COCH2COR‘ ligand on the bimetallic center to produce the carbonyl complex and CH3COR‘ via the enolato−carbonyl complexes [Cp*Ru(CO)(μ2-SPri)2Ru(OCR‘CH2)Cp*]+. In accordance with this mechanism, the reaction of 3a with MeOH afforded [Cp*Ru(μ2-SPri)2{η1:η1-μ2-C(OMe)CHCOOMe}RuCp*][OTf] (7). Complexes 3a and 3b reacted with p-tolylacetylene to form the dinuclear metallacyclic complexes 9a and 9b [Cp*Ru(μ2-SPri)2{η1:η1-μ2-C(Tol)CCCHC(R‘)O}RuCp*][OTf] (9a, R‘ = OMe; 9b, R‘ = Me), respectively. The structures of 3b, 5, 7, and 9a have been determined by X-ray crystallography.

ChemInform Abstract: “Constructive” Creation of a Bimetallic Center via 1,4‐Migration of a Trimethylsilyl Group from Oxygen to Carbon.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

"Constructive" Creation of a Bimetallic Centre via 1,4-Migration of a Trimethylsilyl Group from Oxygen to Carbon

"Constructive" Creation of a Bimetallic Centre via 1,4-Migration of a Trimethylsilyl Group from Oxygen to Carbon

Alkane Transformations on Supported Platinum Catalysts: Part 3: The Stability of Pt/Al2O3(EUROPT-3) and of PtRe/Al2O3(EUROPT-4) during the Hydrogenolysis of Alkanes

The hydrogenolysis of ethane, propane andn-butane has been studied on Pt/Al2O3(EUROPT-3) and PtRe/Al2O3(EUROPT-4) catalysts between 520 and 660 K by a thermal cycling technique using a 10-fold excess of H2. Analogous catalysts having higher metal concentrations have also been examined. With neither ethane nor propane does any progressive deactivation result from repeated thermal cycles, but withn-butane on Pt/Al2O3catalysts initial loss of activity alters product selectivities, specifically by decreasing the probability of breaking the internal C–C bond. After this, they behave reproducibly. With PtRe/Al2O3catalysts, product selectivities shown byn-butane are very different from those given by the Pt/Al2O3catalysts but are unaltered by the somewhat greater initial deactivation; they show some sample-to-sample variation, which is attributed to differences in surface Re concentrations. These results are explained by a model in which sites on low-index planes in very small Pt particles are those that are first inactivated through the formation of strongly held dehydrogenated species; when Re atoms are present they preferentially occupy these sites and the operation of bimetallic centres explains why deeper hydrogenolysis occurs in this case. Reproducible rates ofn-butane hydrogenolysis are obtained with Pt/Al2O3below about 600 K, but not with PtRe/Al2O3; use of short (1 min) reaction pulses overcomes this difficulty. Partially deactivated catalysts can, however, be studied in the continuous-flow mode.

Mutational analysis of the catalytic subunit of muscle protein phosphatase-1.

A mutational analysis of rabbit skeletal muscle protein phosphatase-1 was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli. The selection of the sites to be mutated was based on sequence alignments which showed the existence of a number of invariant residues when eukaroytic Ser/Thr protein phosphatases were compared with bacteriophage phosphatases and adenosinetetraphosphatase [Barton et al. (1995) Eur. J. Biochem. 220, 225-237]. In other studies, it had been shown that PP1 is a metalloprotein [Chu et al. (1996) J. Biol. Chem. 271, 2574-2577], and in this study, we have largely focused on invariant histidine and aspartate residues which may be involved in metal binding. The residues which were mutated were H66, H125, H173, H248, D64, D71, D92, D95, N124, and R96E. The results showed that mutation of H66, H248, D64, and D92 resulted in severe loss of catalytic function. Mutation of D95, N124, and R96 also led to loss of function, while attempts to mutate H125 and H173 led to production of insoluble, inactive proteins. The results of the mutational analysis are consistent with the involvement of conserved His and Asp residues in metal binding, and are discussed in the context of the recently described crystal structure of PP1 [Goldberg et al. (1995) Nature, 376, 745-753], which reveals that PP1 possesses a bimetallic center at the active site. The behavior of the D95, R96, and N124 mutants supports a catalytic mechanism involving nucleophilic attack by a hydroxide ion with H125 functioning as a proton donor to the leaving alcohol group.

EPR studies of wild-type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides: Glu286 is not a bridging ligand in the cytochrome a3-CuB center

Wild-type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides were characterized by EPR spectroscopy. A pH-induced g12 signal, seen previously in mammalian cytochrome oxidase and assigned to the presence of a bridging car☐yl ligand in the bimetallic cytochrome a3-CuB site, is found also in the bacterial enzyme. Mutation of glutamate-286 to glutamine inactivates the enzyme but does not affect this signal, demonstrating that the car☐yl group of this residue is not the bridging ligand. Three mutants, M106Q, located one helix turn below a histidine ligand to cytochrome a, and T352A as well as F391Q, located close to the bimetallic center, are shown to affect dramatically the low-spin heme signal of cytochrome a. These mutants are essentially inactive, suggesting that these three mutations result in alterations to cytochrome a that render the oxidase non-functional.

Kinetics and mechanism for the binding of HCN to cytochrome c oxidase

The kinetics of cyanide binding to cytochrome c oxidase were systematically studied as a function of [HCN], [oxidase], pH, ionic strength, temperature, type and concentration of solubilizing detergent, and monomer-dimer content of oxidase. On the basis of these results a minimum reaction mechanism is proposed in which the spectrally visible rapid and slow cyanide binding reactions are two consecutive first-order reactions, not parallel reactions with different conformers of cytochrome c oxidase. The fast reaction (k'obs) follows saturation type kinetics to form an HCN complex that subsequently undergoes a slow reaction (k'obs). The fast k'obs reaction is independent of ionic strength but is strongly dependent upon pH. Two pK values were evaluated from the bell-shaped rate versus pH profile; one is due to an ionizable group on the protein (pKa = 7.45), while the other is that of HCN (pKHCN = 9.15). Therefore, oxidase is reactive toward HCN only when the group on the protein is unprotonated. The slow k'obs reaction is not a reaction of oxidase with either CN- or HCN; in fact, the product formed by the fast k'obs reaction, the oxidase-HCN complex, still undergoes the slow k" process even if all of the excess KCN is removed. The apparent rate constant of the slower phase (k"obs) is independent of all the variations done in this study, and it probably corresponds to either a slow conformational change in the protein or a change in ligand coordination at one of the metal centers after HCN binds to the bimetallic center of oxidase. Based upon the bell-shaped pH dependence of the fast phase and the pH independence of the slow phase, the mechanism also predicts that a single conformer of cytochrome c oxidase can exhibit either monophasic or biphasic cyanide binding kinetics depending upon the pH. At either very low or very high pH, the two rates become comparable in magnitude, which makes the reaction appear to be monophasic even though both reactions still occur. The amount of monomeric or dimeric oxidase only slightly affects the magnitude of k'obs and k"obs values, and both processes are clearly present in both types of oxidase.

Cytochrome c oxidase: biphasic kinetics result from incomplete reduction of cytochrome a by cytochrome c bound to the high-affinity site.

The electron-transfer kinetics of cytochrome c oxidase were probed by measuring the reduction levels of bound cytochrome c, cytochrome a, and cytochrome a3 during steady-state turnover. Our experimental approach was to measure these reduction levels as a function of (1) the rate of electron input into tightly bound cytochrome c by varying the concentration of TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) and/or cytochrome c and (2) the rate of electron efflux out of cytochrome a (true Kcat) by changing the detergent surrounding cytochrome c oxidase. In most detergent environments, the rate of electron input into cytochrome c is not faster than the rate of electron efflux from cytochrome a. The relatively slow rate of electron input results in incomplete reduction of both cytochrome a and cytochrome c bound a the high-affinity site unless Kcat is very slow. When the high-affinity site is saturated with cytochrome c, the steady-state reduction level of cytochrome a defines Vmax,1, which is the maximum velocity of the high-affinity phase. The remaining fractional oxidation level of cytochrome a determines Vmax,2, the maximum velocity of the low-affinity phase. Therefore, it is the sum Vmax,1 + Vmax,2 which defines the maximum rate of electron transfer between cytochrome a and the bimetallic center, i.e., Kcat. We also were able to evaluate the true Kcat of cytochrome c oxidase in each detergent environment directly from the steady-state reduction levels without any of the complications introduced by the analysis of the polarographic kinetic data.(ABSTRACT TRUNCATED AT 250 WORDS)