Cytochrome cd1, Reductive Activation and Kinetic Analysis of a Multifunctional Respiratory Enzyme

Richter, Carsten D.; Allen, James W. A.; Higham, Christopher W.; Koppenhöfer, Alrik; Zajicek, Richard S.; Watmough, Nicholas J.; Ferguson, Stuart J.

doi:10.1074/jbc.m108944200

Cited by 54 publications

(84 citation statements)

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“…Pseudoazurin (PAz) is proposed to be one of the electron carriers in the periplasm of the bacteria, shuttling electrons between the cytochrome bc 1 complex and several periplasmic enzymes involved in denitrification as well as CCP [14,15]. Another small redox protein, cytochrome c 550 , has also been identified as the electron donor to these enzymes [14,15].…”

Section: Introductionmentioning

confidence: 99%

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer

et al. 2007

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This work reports the direct electrochemistry of Paracoccus pantotrophus pseudoazurin and the mediated catalysis of cytochrome c peroxidase from the same organism. The voltammetric behaviour was examined at a gold membrane electrode, and the studies were performed in the presence of calcium to enable the peroxidase activation. A formal reduction potential, E 0 ¢, of 230 ± 5 mV was determined for pseudoazurin at pH 7.0. Its voltammetric signal presented a pH dependence, defined by pK values of 6.5 and 10.5 in the oxidised state and 7.2 in the reduced state, and was constant up to 1 M NaCl. This small copper protein was shown to be competent as an electron donor to cytochrome c peroxidase and the kinetics of intermolecular electron transfer was analysed. A second-order rate constant of 1.4 ± 0.2 · 10 5 M -1 s -1 was determined at 0 M NaCl. This parameter has a maximum at 0.3 M NaCl and is pH-independent between pH 5 and 9.

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Section: Introductionmentioning

confidence: 99%

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer

et al. 2007

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“…The switch to His/Met ligation significantly raises the reduction potential of heme c, more closely matching the donor proteins cytochrome c 550 and pseudoazurin (8 -13). This led to the speculation that this facilitates electron transfer (8) but a low heme c potential is not strictly an obstacle because the overall electron transfer from donors to NO 2 Ϫ is thermodynamically favorable (13,14). The purpose of the ligand switch is therefore unclear.…”

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confidence: 99%

“…Until this reversion has occurred, the enzyme is capable of NO 2 Ϫ reduction using the physiological electron donor pseudoazurin (11). It is reported that for full activity, oxidized Pp cd 1 requires preactivation by chemical reduction (11,13). Consequently, His/Met ligated heme c is presumed to be a prerequisite of activity, and it has been proposed that the oxidized enzyme is a resting form bypassed in catalysis as the enzyme cycles rapidly compared with the rate of reversion to the His/His form (13,26).…”

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confidence: 99%

“…It is reported that for full activity, oxidized Pp cd 1 requires preactivation by chemical reduction (11,13). Consequently, His/Met ligated heme c is presumed to be a prerequisite of activity, and it has been proposed that the oxidized enzyme is a resting form bypassed in catalysis as the enzyme cycles rapidly compared with the rate of reversion to the His/His form (13,26). The oxidized state would therefore not exist in vivo without an appropriate reactivation mechanism (8,12,26).…”

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confidence: 99%

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Activation of the Cytochrome cd1 Nitrite Reductase from Paracoccus pantotrophus

Wonderen

Knight

Oganesyan

et al. 2007

Journal of Biological Chemistry

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Cytochromes cd 1 are dimeric bacterial nitrite reductases, which contain two hemes per monomer. On reduction of both hemes, the distal ligand of heme d 1 dissociates, creating a vacant coordination site accessible to substrate. Heme c, which transfers electrons from donor proteins into the active site, has histidine/methionine ligands except in the oxidized enzyme from Paracoccus pantotrophus where both ligands are histidine. During reduction of this enzyme, Tyr 25 dissociates from the distal side of heme d 1 , and one heme c ligand is replaced by methionine. Activity is associated with histidine/methionine coordination at heme c, and it is believed that P. pantotrophus cytochrome cd 1 is unreactive toward substrate without reductive activation. However, we report here that the oxidized enzyme will react with nitrite to yield a novel species in which heme d 1 is EPR-silent. Magnetic circular dichroism studies indicate that heme d 1 is low-spin Fe III but EPR-silent as a result of spin coupling to a radical species formed during the reaction with nitrite. This reaction drives the switch to histidine/methionine ligation at Fe III heme c. Thus the enzyme is activated by exposure to its physiological substrate without the necessity of passing through the reduced state. This reactivity toward nitrite is also observed for oxidized cytochrome cd 1 from Pseudomonas stutzeri suggesting a more general involvement of the EPR-silent Fe III heme d 1 species in nitrite reduction.

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“…In nitrite reductase assays, turnover occurs at approximately 10 2 s À1 [37]. Fourier transform infrared (FTIR) studies show that the initial reaction with nitrite proceeds with k > 500 s À1 and a subsequent rearrangement occurs in approximately 40 ms [38].…”

Section: Assessing the Efficiency Of Reagent Injection And Stirring Mmentioning

confidence: 99%

Electrochemical titrations and reaction time courses monitored in situ by magnetic circular dichroism spectroscopy

Bradley

Butt

Cheesman

2011

Analytical Biochemistry

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a b s t r a c tMagnetic circular dichroism (MCD) spectra, at ultraviolet-visible or near-infrared wavelengths (185-2000 nm), contain the same transitions observed in conventional absorbance spectroscopy, but their bisignate nature and more stringent selection rules provide greatly enhanced resolution. Thus, they have proved to be invaluable in the study of many transition metal-containing proteins. For mainly technical reasons, MCD has been limited almost exclusively to the measurement of static samples. But the ability to employ the resolving power of MCD to follow changes at transition metal sites would be a potentially significant advance. We describe here the development of a cuvette holder that allows reagent injection and sample mixing within the 50-mm-diameter ambient temperature bore of an energized superconducting solenoid. This has allowed us, for the first time, to monitor time-resolved MCD resulting from in situ chemical manipulation of a metalloprotein sample. Furthermore, we report the parallel development of an electrochemical cell using a three-electrode configuration with physically separated working and counter electrodes, allowing true potentiometric titration to be performed within the bore of the MCD solenoid.Ó 2011 Elsevier Inc. All rights reserved.Magnetic circular dichroism (MCD) 1 spectroscopy, at ultraviolet-visible and near-infrared wavelengths (185-2000 nm), has proved to be invaluable in the study of metalloproteins containing cofactors such as heme [1][2][3], non-heme iron [4], iron-sulfur clusters [5][6][7][8], cobalt [9,10], nickel [11][12][13], and copper [14,15]. The technique measures the apparent circular dichroism (CD) induced by a magnetic field [16]. Despite similarities in the instrumentation used, the observation of MCD is not dependent on the chirality of the protein; the method is equally applicable to racemic model complexes [17]. The magnetic field will always induce signals across wavelengths at which the substance absorbs. Thus, MCD spectra contain the same electronic transitions observed in conventional absorbance spectroscopy, but the bisignate nature of the spectrum provides enhanced resolution and greater detail. This spectral detail offers an unmatched fingerprinting capability that can, for example, identify the spin and oxidation states of heme groups [1] and distinguish among the variety of iron-sulfur centers found in biological molecules [18][19][20].Metalloprotein MCD can be measured at ambient or cryogenic temperatures. The latter requires adulteration with glassing agents [16] but has generally been preferred because MCD intensity from paramagnetic centers increases dramatically at low temperature. Thus, most metalloprotein MCD reported has been measured in glasses at temperatures of approximately 4.2 K. Hemoproteins represent the significant exception, giving rise to appreciable MCD at high temperatures [1]. Thus, ambient temperature MCD is used to diagnose spin state, oxidation state, and (in the case of low-spin Fe(III) hemes) axial ligation [21].As ...

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Cytochrome cd1, Reductive Activation and Kinetic Analysis of a Multifunctional Respiratory Enzyme

Cited by 54 publications

References 44 publications

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer

Activation of the Cytochrome cd1 Nitrite Reductase from Paracoccus pantotrophus

Electrochemical titrations and reaction time courses monitored in situ by magnetic circular dichroism spectroscopy

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