Functional role of heme ligation in cytochrome c. Effects of replacement of methionine 80 with natural and non-natural residues by semisynthesis.

Wallace, Carmichael J. A.; Clark‐Lewis, Ian

doi:10.1016/s0021-9258(19)50604-4

Cited by 118 publications

(70 citation statements)

References 35 publications

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“…The electronic absorption spectra of ferric Y67R and Y67R/M80A at pH 7.4 are similar (Figures A and A). The Y67R/M80A variant does not have a split Q-band, which is apparent for the hydroxide-ligated M80A, − suggesting that the heme ligand in this variant is likely not a hydroxide. This conclusion is further supported by NMR spectra (Figure A).…”

Section: Resultsmentioning

confidence: 95%

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Remote Perturbations in Tertiary Contacts Trigger Ligation of Lysine to the Heme Iron in Cytochrome c

Shin

Pletneva

2017

Biochemistry

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Perturbations in protein structure define the mechanism of allosteric regulation and biological information transfer. In cytochrome c (cyt c), Met80 ligation to the heme iron is critical for the protein’s electron-transfer (ET) function in oxidative phosphorylation and for suppressing its peroxidase activity in apoptosis. The hard base Lys is a better match for the hard ferric iron than the soft base Met, suggesting the key role of the protein scaffold in favoring Met ligation. To probe the role of the protein structure in maintenance of Met ligation, mutations T49V and Y67R/M80A were designed to disrupt hydrogen bonding and packing of the heme coordination loop, respectively. Electronic absorption, NMR, and EPR spectra reveal that ferric forms of both variants are Lys-ligated at neutral pH. A minor change in the tertiary contacts in T49V, away from the heme coordination loop, appears to be sufficient to execute a change in ligation, suggesting a cross-talk between the different regions of the protein structure and a possibility of built-in conformational switches in cyt c. Analyses of thermodynamic stability, kinetics of Lys binding and dissociation and the pH-dependent changes in ligation provide detailed characterization of the Lys coordination in these variants and relate these properties to the extent of structural perturbations. The findings emphasize the importance of the hydrogen-bonding network in controlling the native Met80 ligation to the heme iron.

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Section: Resultsmentioning

confidence: 95%

“… a In this work, potentials were determined from spectroelectrochemistry experiments performed at 22 ± 2 °C. b From ref . c From ref . d Reductive (oxidative) direction. e From ref . …”

Section: Resultsmentioning

confidence: 99%

Remote Perturbations in Tertiary Contacts Trigger Ligation of Lysine to the Heme Iron in Cytochrome c

Shin

Pletneva

2017

Biochemistry

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“…Lysine–heme coordination has long been studied in the context of alternative conformations of WT and variant c cytochromes. ,,,− Unlike in WT cytochrome, replacing the axial methionine with a lysine by mutagenesis can generate His–Lys complexes that persist in both the ferric and ferrous states. − In a dramatic example of ligand competition, wild-type lysines located in a flexible Ω-loop at positions 72, 73, and 79 can also replace Met80 as an axial ligand to the ferric heme at alkaline pH or high temperature. − ,− 1 H NMR exchange studies on the alkaline isomerization (His–Fe III –Met ⇆ His–Fe III –Lys) of ferric horse cytochrome c indicate that at 300 K, replacement of methionine with lysine is independent of pH ( k f = 4.0 ± 0.6 s –1 ) but that the reverse process ( k r ) is accelerated from 3 s –1 at pH* 10 to 25 s –1 at pH* 8.5 . However, the reverse rate constant determined by NMR is significantly faster than that determined via pH-jump stopped-flow methods ( k f = 6.0 s –1 and k r = 0.05 s –1 ) .…”

Section: Discussionmentioning

confidence: 99%

Dynamics of Lysine as a Heme Axial Ligand: NMR Analysis of the Chlamydomonas reinhardtii Hemoglobin THB1

2017

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Nitrate metabolism in Chlamydomonas reinhardtii involves THB1, a monomeric hemoglobin thought to function as a nitric oxide dioxygenase (NOD). NOD activity requires dioxygen and nitric oxide binding followed by a one-electron oxidation of the heme iron and nitrate release. Unlike pentacoordinate flavohemoglobins, which are efficient NODs, THB1 uses two iron axial ligands: the conserved proximal histidine and a distal lysine (Lys53). As a ligand in both the oxidized (ferric) and reduced (ferrous) states, Lys53 is expected to lower the reorganization energy associated with electron transfer and therefore facilitate reduction of the ferric enzyme. In ferrous THB1, however, Lys53 must be displaced for substrate binding. To characterize Lys53 dynamics, THB1 was studied at various pH, temperatures, and pressures by NMR spectroscopy. Structural information indicates that the protein fold and Lys53 environment are independent of the oxidation state. High-pressure NMR experiments provided evidence that displacement of Lys53 occurs through fast equilibrium (∼3-4 × 10 s at 1 bar, 298 K) with a low-population intermediate in which Lys53 is neutral and decoordinated. Once decoordinated, Lys53 is able to orient toward solvent and become protonated. The global lysine decoordination/reorientation/protonation processes measured by N-exchange spectroscopy are slow on the chemical shift time scale (10-10 s at pH ≈ 6.5, 298 K) in both iron redox states. Thus, reorientation/protonation steps in ferrous THB1 appear to present a significant barrier for dioxygen binding, and consequently, NOD turnover. The results illustrate the role of distal ligand dynamics in regulating the kinetics of multistep heme redox reactions.

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“…Both ligation schemes free up the sixth heme coordination site for diatomic ligand binding when reduced, favoring mono-His ligation in the ferrous state. 42 Two examples also exist of oxyferrous states in engineered variants of cytochrome c (horse heart cytochrome c, S. cerevisiae iso-1cytochrome c) 22,23,27 where the distal heme-ligating methionine ligands are replaced with alanine, freeing the sixth coordination site for ligand binding. The oxyferrous state is exceptionally long-lived in the engineered iso-1-cytochrome c, 27 exhibiting an autoxidation rate that is signicantly lower than that of myoglobin.…”

Section: Discussionmentioning

confidence: 99%

“…The spectral changes observed on binding CO and O 2 are similar to those observed 6 for the corresponding heme B-bound species of 1 and the oxyferrous absorbance spectrum is consistent with the oxyferrous spectra of the few natural and engineered c-type cytochromes that can undergo reversible oxygen binding (ESI, Table S2 †). [22][23][24] The lifetime of the oxyferrous species of 2 is similar to those observed 6 for 1 and 1.5 (1, t 1/2 ¼ 9.4 seconds; 1.5, t 1/2 ¼ 10 seconds; ESI, Table S3 †), though autoxidation at 15 C occurs less rapidly (t 1/2 ¼ 15 seconds), indicating a slight stabilization of the oxyferrous species on switching between hemes B and C. This rate is signicantly more rapid than the autoxidation rates of the natural globins 25,26 and the natural and engineered c-type cytochromes, 27,28 most likely due to the relatively low reduction potential of the hemes in 2 and the other oxygen-binding maquettes, 6 but it is within the range of autoxidation rates exhibited by the oxygen binding and activating cytochromes P450 (ESI, Table S3 †). [29][30][31] Physical chemistry of mixed heme C/B maquettes Titration of one equivalent of heme B into 2 creates a mixed heme C/B maquette (Fig.…”

Section: In Vivo Cofactor Incorporationmentioning

confidence: 99%

Constructing a man-made c-type cytochrome maquette in vivo: electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette.

et al. 2014

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The successful use of man-made proteins to advance synthetic biology requires both the fabrication of functional artificial proteins in a living environment, and the ability of these proteins to interact productively with other proteins and substrates in that environment. Proteins made by the maquette method integrate sophisticated oxidoreductase function into evolutionarily naive, non-computationally designed protein constructs with sequences that are entirely unrelated to any natural protein. Nevertheless, we show here that we can efficiently interface with the natural cellular machinery that covalently incorporates heme into natural cytochromes c to produce in vivo an artificial c-type cytochrome maquette. Furthermore, this c-type cytochrome maquette is designed with a displaceable histidine heme ligand that opens to allow functional oxygen binding, the primary event in more sophisticated functions ranging from oxygen storage and transport to catalytic hydroxylation. To exploit the range of functions that comes from the freedom to bind a variety of redox cofactors within a single maquette framework, this c-type cytochrome maquette is designed with a second, non-heme C, tetrapyrrole binding site, enabling the construction of an elementary electron transport chain, and when the heme C iron is replaced with zinc to create a Zn porphyrin, a light-activatable artificial redox protein. The work we describe here represents a major advance in de novo protein design, offering a robust platform for new c-type heme based oxidoreductase designs and an equally important proof-of-principle that cofactor-equipped man-made proteins can be expressed in living cells, paving the way for constructing functionally useful man-made proteins in vivo.

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Functional role of heme ligation in cytochrome c. Effects of replacement of methionine 80 with natural and non-natural residues by semisynthesis.

Cited by 118 publications

References 35 publications

Remote Perturbations in Tertiary Contacts Trigger Ligation of Lysine to the Heme Iron in Cytochrome c

Remote Perturbations in Tertiary Contacts Trigger Ligation of Lysine to the Heme Iron in Cytochrome c

Dynamics of Lysine as a Heme Axial Ligand: NMR Analysis of the Chlamydomonas reinhardtii Hemoglobin THB1

Constructing a man-made c-type cytochrome maquette in vivo: electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette.

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