Location of the heme groups in cytochrome cd1 oxidase from Pseudomonas aeruginosa

Mitra, Sekhar; Bersohn, R.

doi:10.1021/bi00555a015

Cited by 26 publications

(8 citation statements)

References 16 publications

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“…The presence of mutually unfavourable interactions between the two sites binding reduced cytochrome c-551 may tentatively be correlated to the limited structural information available on cytochrome oxidase, which is a symmetrical dimer where both the haem c pair and the haem d pair are located close to each other (Takano et al, 1979). Moreover, fluorescence energy-transfer experiments have indicated that the four haem groups are segregated at one extreme of the dimeric structure and that two large protein domains in the dimer are quite distant from the active site (Mitra & Bershon, 1980). It is not at all unreasonable, therefore, to assume that binding of a reduced cytochrome c-55 1 molecule to one of the two subunits of the dimer may limit, by steric hindrance and/or electrostatic effects, binding to the other subunit.…”

Section: Discussionmentioning

confidence: 90%

Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study

Tordi¹,

Silvestrini²,

Colosimo³

et al. 1985

Biochemical Journal

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The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a 'half-hearted' behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.

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Section: Discussionmentioning

confidence: 90%

Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study

Tordi¹,

Silvestrini²,

Colosimo³

et al. 1985

Biochemical Journal

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“…Alcaligenes azurin was prepared at 40C by the procedure used for preparing respiratory chain proteins from P. aeruginosa (14) …”

Section: Methodsmentioning

confidence: 99%

Proton NMR of the histidines of azurin from Alcaligenes faecalis: linkage of histidine-35 with redox kinetics.

Mitra¹,

Bersohn²

1982

Proc. Natl. Acad. Sci. U.S.A.

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On the basis of redox kinetic studies, Rosen and Pecht [Rosen, P. & Pecht, I. (1976) Biochem. 120,[339][340][341][342][343][344] observed that the azurin from the bacterium Alcaligenesfaecalis shows no such slowly attained equilibrium between two forms. Therefore, a 1H NMR study was carried out on this azurin with emphasis on the downfield region. A resonance was found at 7.95 ppm downfield that does not move with pH, is not seen in the oxidized protein, has the same pseudocontact shift in the Co(II) derivative as the C-2 proton of histidine-35 has in the Co(E) derivative of P. aeruginosa azurin, and, in the apoprotein, exhibits a typical protonation shift downfield at pH <5. Therefore, this resonance is assigned to the C-2 proton of histidine-35. The crystal structure ofP. aeruginosa azurin shows that at pH 7 the imidazole side chain ofhistidine-35 is in a crevice within the protein, where its ring is adjacent and parallel to that ofhistidine-47, a copper ligand. The preceding observations combined with others show that the kinetics of some redox reactions involving azurin depend on the position of histidine-35. The implication is that there is a pathway for electron transport to the copper atom involving passage through histidine-35.An azurin is a deep blue, low molecular weight (Mr 14,000), bacterial protein that contains one Cu(II) per molecule. The crystal structure of one of them, the azurin from Pseudomonas aeruginosa has been solved (1,2), and the copper ligands are nitrogen atoms from His-46 and His-117 and sulfur atoms from Cys-112 and Met-121. These four amino acids (and many others) are invariant in all of the azurins with sequences determined so far (3). Indeed, the deep blue color has been convincingly explained as being due to a charge-transfer transition from the cysteinate sulfur to Cu(II) (4, 5).The function of azurins in bacteria has not yet been elucidated, but a generous hint is furnished by the fast rate (6) of electron transfer between P. aeruginosa azurin and cytochrome c551, another small protein found in the same bacterium. When the temperature is suddenly raised, the redox equilibrium shifts toward reduced azurin with a fast time constant and then shifts back again with a slow time constant (7,8). Thus, there are two interesting aspects to the reaction. One is the rapid electron transfer (k 106 M -1sec 1) between two proteins, both ofwhose metal ions are well shielded from the solvent. The other is the slow interconversion of two conformers of the same protein, reduced azurin, one ofwhich is much more reactive in electron transfer than the other. Previous NMR (9-11) and temperaturejump studies (unpublished data) have shown that the slowly attained protonation-deprotonation equilibrium of the imidazole side chains of His-35 may be the slow step in the redox process. Normally the exchange time near neutral pH is short (<1 psec). The long exchange time of 0O. 1 sec actually found shows that the protonation must be accompanied by a local conformation change that alters the ef...

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“…A definite role for azurin has yet to be established (Henry & Bessiers, 1984). Azurins will react rapidly with cytochrome c-551 and with cytochrome cd, the soluble dimeric terminal oxidase (Gorin et al, 1983; Mitra & Bersohn, 1980;Silvestrini et al, 1981;Takano et al, 1979; Ugurbil et al, 1985). We have chosen to study holoand apoazurins of Pseudomonas aeruginosa (azurin Pae),1 Alcali genes faecalis (azurin Afe), and Alcaligenes denitrificans (azurin Ade).…”

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confidence: 99%

Internal motion and electron transfer in proteins: a picosecond fluorescence study of three homologous azurins

Petrich¹,

Longworth²,

Fleming³

1987

Biochemistry

117

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We have carried out a picosecond fluorescence study of holo- and apoazurins of Pseudomonas aeruginosa (azurin Pae), Alcaligenes faecilis (azurin Afe), and Alcaligenes denitrificans (azurin Ade). Azurin Pae contains a single, buried tryptophyl residue; azurin Afe, a single surface tryptophyl residue; and azurin Ade, tryptophyl residues in both environments. From anisotropy measurements we conclude that the interiors of azurins Pae and Ade are not mobile enough to enable motion of the indole ring on a nanosecond time scale. The exposed tryptophans in azurins Afe and Ade show considerable mobility on a few hundred picosecond time scale. The quenching of tryptophan fluorescence observed in the holoproteins is interpreted in terms of electron transfer from excited-state tryptophan to Cu(II). The observed rates are near the maximum predicted by Marcus theory for the separation of donor and acceptor. The involvement of protein matrix and donor mobility for electron transfer is discussed. The two single-tryptophan-containing proteins enable the more complex fluorescence behavior of the two tryptophans of azurin Ade to be understood. The single-exponential fluorescence decay observed for azurin Pae and the nonexponential fluorescence decay observed for azurin Afe are discussed in terms of current models for tryptophan photophysics.

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Location of the heme groups in cytochrome cd1 oxidase from Pseudomonas aeruginosa

Cited by 26 publications

References 16 publications

Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study

Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study

Proton NMR of the histidines of azurin from Alcaligenes faecalis: linkage of histidine-35 with redox kinetics.

Internal motion and electron transfer in proteins: a picosecond fluorescence study of three homologous azurins

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