Isolation and characterization of cytochrome c550 from the methylamine‐oxidizing electron‐transport chain of Thiobacillus versutus

Lommen, Arjen; Ratsma, Age; Bijlsma, Niels; Canters, Gerard W.; Wielink, John E. van; Frank, Johannes; Beeumen, Jozef Van

doi:10.1111/j.1432-1033.1990.tb19272.x

Cited by 33 publications

(49 citation statements)

References 37 publications

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“…Such a protonation does not induce significant spectral changes, and it can probably be attributed to the protonation of a heme propionate(s) (14), which have already been suggested to be responsible for the "redox Bohr effect" in several cytochromes (15)(16)(17)(18). Therefore, the behavior reported in Fig.…”

Section: Figmentioning

confidence: 75%

pH Dependence of Structural and Functional Properties of Oxidized Cytochrome c" from Methylophilus methylotrophus

Coletta¹,

Costa²,

Sanctis³

et al. 1997

Journal of Biological Chemistry

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Cytochrome c؆ from Methylophilus methylotrophus is an unusual monoheme protein that undergoes a major redox-linked change in the heme arrangement: one of the two axial histidines bound to the iron in the oxidized form is detached upon reduction and a proton is taken up. The kinetics of reduction by sodium dithionite and the spectroscopic properties of the oxidized cytochrome c؆ have been investigated over the pH range between 1.4 and 10.0. The rate of reduction displays proton-linked transitions of pK a Х 5.5 and 2.4, and a spectroscopic transition with a pK a Х 2.4 is also observed. The protein displays a complete reversibility after exposure to low pH, and both electronic absorption and resonance Raman spectroscopic properties suggest that the transition at lower pH brings about a drastic change in the heme coordination geometry. Circular dichroism spectra indicate that over the same proton-linked transition, the protein undergoes a marked decrease (ϳ60%) of the ␣-helical content toward a random coil arrangement, which is recovered upon increasing the ionic strength. The structural change at low pH is linked to a concerted two-proton transition, suggesting the detachment and protonation of axial histidine(s). Such kinetic and spectroscopic features along with the remarkable capacity of this protein to recover its native structure after exposure to extremely low pH values makes it a promising model for studying folding processes and stability in heme proteins.Cytochrome cЉ from the obligate methylotroph Methylophilus methylotrophus is a soluble monoheme protein of ϳ15 kDa, which displays a redox-linked spin state transition from a low spin state in the oxidized form to a high spin state in the reduced form (1). The two axial ligands are histidines in the oxidized form, one of which is detached from heme upon reduction of the iron atom (2). Cytochrome cЉ is a unique example of a heme-c protein with bis-histidine coordination and spectroscopic features similar to those observed in model compounds where axial ligand planes are forced into a perpendicular orientation by steric constraints (3). NMR studies of the heme pocket have shown that it is quite stable at neutral pH, with low amide proton exchange rates and one of the heme propionates largely exposed at the surface, whereas the other one is buried in the protein (1). Attempts to crystallize the protein have been unsuccessful to date. The N-terminal half of the amino acid sequence has been determined, and it displays no significant similarity with sequences from any other protein (4).The midpoint redox potential of cytochrome cЉ has a strong pH dependence (i.e. a redox Bohr effect) over the range between pH 4.0 and 10.0 (4). NMR spectroscopy shows that some of the methyl resonances in the oxidized form display a shift as large as 3 ppm over the same range. The pH dependence of the midpoint redox potential has been analyzed in terms of a model that considers two ionizing groups with pK a values that change with the redox state of the protein (1, 4). It was conc...

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Section: Figmentioning

confidence: 75%

pH Dependence of Structural and Functional Properties of Oxidized Cytochrome c" from Methylophilus methylotrophus

Coletta¹,

Costa²,

Sanctis³

et al. 1997

Journal of Biological Chemistry

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“…This is also the case for the alkaline transition in cyt c-550, where a single coordinating lysine species is observed by NMR spectroscopy. [55] The effect of the K99E mutation on unfolding at pH 7.0…”

Section: Dmentioning

confidence: 99%

The Effects of Ligand Exchange and Mobility on the Peroxidase Activity of a Bacterial Cytochrome c upon Unfolding

et al. 2005

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The effect on the heme environment upon unfolding Paracoccus versutus ferricytochrome c-550 and two site-directed variants, K99E and H118Q, has been assessed through a combination of peroxidase activity increase and one-dimensional NMR spectroscopy. At pH 4.5, the data are consistent with a low- to high-spin heme transition, with the K99E mutation resulting in a protein with increased peroxidase activity in the absence of or at low concentrations of denaturant. Furthermore, the mobility of the polypeptide chain at pH 4.5 for the wild-type protein has been monitored in the absence and presence of denaturant through heteronuclear NMR experiments. The results are discussed in terms of local stability differences between bacterial and mitochondrial cytochromes c that are inferred from peroxidase activity assays. At pH 7.0, a mixture of misligated heme states arising from protein-based ligands assigned to lysine and histidine is detected. At low denaturant concentrations, these partially unfolded misligated heme forms inhibit the peroxidase activity. Data from the K99E mutation at pH 7.0 indicate that K99 is not involved in heme misligation, whereas histidine coordination is proven by the data from the H118Q variant.

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“…Ongoing studies of the mechanism of the electron transfer between the various reaction partners in the chain have shown among others, that the redox activity of the amicyanin in vitro is regulated by pH [6]. The terminal oxidase in the redox chain is an aa3-type cytochrome oxidase [1, 21. The link between the oxidase and the amicyanin is presumably formed by cytochrome cS5,, [l, 201, a single heme containing class I c-type cytochrome with a molecular mass of 15 kDa, which recently has been characterised extensively [4].…”

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confidence: 99%

Cloning, sequencing and expression studies of the genes encoding amicyanin and the β‐subunit of methylamine dehydrogenase from Thiobacillus versutus

Ubbink

Kleef

Kleinjan

et al. 1991

European Journal of Biochemistry

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The genes encoding amicyanin and the P-subunit of methylamine dehydrogenase (MADH) from Thiohacillus versutus have been cloned and sequenced. The organization of these genes makes it likely that they are coordinately expressed and it supports earlier findings that the blue copper protein amicyanin is involved in electron transport from methylamine to oxygen. The amino acid sequence deduced from the nucleotide sequence of the amicyaninencoding gene is in agreement with the published protein sequence. The gene codes for a pre-protein with a 25-amino-acid-long signal peptide. The amicyanin gene could be expressed efficiently in Escherichia coli. The protein was extracted with the periplasmic fraction, indicating that pre-amicyanin is translocated across the inner membrane of E. coli. Sequence studies on the purified P-subunit of MADH confirm the amino acid sequence deduced from the nucleotide sequence of the corresponding gene. The latter codes for a pre-protein with an unusually long (56 amino acids) leader peptide. The sequencing results strongly suggest that pyrroloquinoline quinone (PQQ) or pro-PQQ is not the co-factor of MADH.When Thiobacillus versutus is offered methylamine as its sole source of carbon, nitrogen and energy, it synthesises a redox chain which converts the amine into the aldehyde and which transports the electrons generated in the methylamine conversion eventually to oxygen as the final electron acceptor [1]. Because the redox chain is short and the constituting proteins are relatively easy to manipulate, there is currently strong interest in the physiology, enzymology and mode of operation of this chain [l -91. Although the composition of this chain has not yet been established with absolute certainty, there is agreement [l, 3, 5, 10, 111 that the first two proteins in the redox path are: (a) methylamine dehydrogenase (MADH), an a2P2 dimer with a molecular mass of 123 kDa and a quinone compound as a prosthetic group [12, 131 and (b) amicyanin, a type I copper protein with a molecular mass of 11.7 kDa [ 14, 151. Crystals of MADH have been analysed with X-ray diffraction techniques but the,interpretation of the

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Isolation and characterization of cytochrome c₅₅₀ from the methylamine‐oxidizing electron‐transport chain of Thiobacillus versutus

Cited by 33 publications

References 37 publications

pH Dependence of Structural and Functional Properties of Oxidized Cytochrome c" from Methylophilus methylotrophus

pH Dependence of Structural and Functional Properties of Oxidized Cytochrome c" from Methylophilus methylotrophus

The Effects of Ligand Exchange and Mobility on the Peroxidase Activity of a Bacterial Cytochrome c upon Unfolding

Cloning, sequencing and expression studies of the genes encoding amicyanin and the β‐subunit of methylamine dehydrogenase from Thiobacillus versutus

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