Electrostatic Environment of Hemes in Proteins:  p<i>K</i><sub>a</sub>s of Hydroxyl Ligands

Song, Yifan; Mao, Junjun; Gunner, M. R.

doi:10.1021/bi052182l

Cited by 30 publications

(36 citation statements)

References 86 publications

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“…Conformers are built for waterheme a 3 , hydroxyl-heme a 3 , water-Cu B , and hydroxylCu B . On heme a 3 , the Fe-O bond is oriented perpendicular to the porphyrin plane, and the bond length is 1.95 Å with square bipyramidal geometry (53). The oxygen position for Cu B is determined by DFT optimization calculations using the B3LYP method (77) and LANL2DZ basis set (78) for an isolated cupric hydroxyl-3His-Cu complex.…”

Section: Methodsmentioning

confidence: 99%

“…A +0.3 charge is placed on the formyl group C and -0.3 on the electron-withdrawing formyl group of the a-type hemes. This simple metal-centered charge set has been used successfully in heme benchmark calculations for bis-His and His-aquo hemes (25,27,53).…”

Section: Methodsmentioning

confidence: 99%

“…On the basis of a study of aquo-heme pK a s and E m s in various proteins (53), the Poisson-Boltzmann continuum electrostatics calculations are found to overestimate the interactions of ferric heme a 3 with Cu B . That study compared the energies of a positive charge near a ferric aquo-heme in a vacuum calculated with DFT and with Coulomb's law.…”

Section: Methodsmentioning

confidence: 99%

“…While interactions with negative charges, with charges in the heme plane, such as the propionic acids, and with distant charges, such as heme a, are well represented by Coulomb's law, when a charge is near the face of the heme, the interactions are overestimated. Interactions with groups close to the heme also need correction in the benchmark calculations of the aquo-heme pK a s in sperm whale and Aplysia myoglobin, hemoglobin I, heme oxygenase 1, and horseradish peroxidase (53).…”

Section: Methodsmentioning

confidence: 99%

“…Given measured aqueous pK a s and E m s (pK a,sol and E m,sol ) for acidic and basic groups and redox cofactors, MCCE provides these values in the protein. The program is able to calculate pK a s for amino acids (51), heme propionates (27), hydroxides ligated to hemes (53), and E m s for hemes in a variety of cytochromes (25,27) through benchmark calculations on simpler proteins. Prior studies on photosynthetic reaction centers (52,54,55), bacteriorhodopsin (56), and quinol:fumarate reductase (57) show that MCCE provides a good match to experimental values for cofactor E m s and both cofactor and amino acid pK a s in large transmembrane proteins.…”

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confidence: 99%

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Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

2006

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Cytochrome c oxidase is a transmembrane proton pump that builds an electrochemical gradient using chemical energy from the reduction of O 2 . Ionization states of all residues were calculated with Multi-Conformation Continuum Electrostatics (MCCE) in seven anaerobic oxidase redox states ranging from fully oxidized to fully reduced. One long-standing problem is how proton uptake is coupled to the reduction of the active site binuclear center (BNC). The BNC has two cofactors: heme a 3 and Cu B . If the protein needs to maintain electroneutrality, then 2 protons will be bound when the BNC is reduced by 2 electrons in the reductive half of the reaction cycle. The effective pK a s of ionizable residues around the BNC are evaluated in Rhodobacter sphaeroides cytochrome c oxidase. At pH 7, only a hydroxide coordinated to Cu B shifts its pK a from below 7 to above 7 and so picks up a proton when heme a 3 and Cu B are reduced. Glu I-286, Tyr I-288, His I-334, and a second hydroxide on heme a 3 all have pK a s above 7 in all redox states, although they have only 1.6-3.5 ∆pK units energy cost for deprotonation. Thus, at equilibrium, they are protonated and cannot serve as proton acceptors. The propionic acids near the BNC are deprotonated with pK a s well below 7. They are well stabilized in their anionic state and do not bind a proton upon BNC reduction. This suggests that electroneutrality in the BNC is not maintained during the anaerobic reduction. Proton uptake on reduction of Cu A , heme a, heme a 3 , and Cu B shows ≈2.5 protons bound per 4 electrons, in agreement with prior experiments. One proton is bound by a hydroxyl group in the BNC and the rest to groups far from the BNC. The electrochemical midpoint potential (E m ) of heme a is calculated in the fully oxidized protein and with 1 or 2 electrons in the BNC. The E m of heme a shifts down when the BNC is reduced, which agrees with prior experiments. If the BNC reduction is electroneutral, then the heme a E m is independent of the BNC redox state.Heme-copper oxidases are the terminal electron acceptors in anaerobic organisms. These transmembrane proteins reduce dioxygen and convert the released chemical energy into an electrochemical gradient, across the eukaryotic mitochondrial membrane or the bacterial cell membrane (1-4). Cytochrome c oxidases are the most prevalent hemecopper oxidases. In this protein 4 electrons, provided by 4 cytochromes c, are used to reduce dioxygen to water. The 4 protons needed to make water are taken from the negative cytoplasmic side of the membrane, adding to the electrochemical gradient. Four additional protons are pumped across the membrane (5). Thus, each dioxygen molecule reduced by cytochrome c oxidase is coupled to the transfer of 8 charges across the membrane.The first step of electron transfer is from cytochrome c to Cu A , a dicopper center in subunit II which extends beyond the membrane on the proton release side of the protein (Figure 1). After receiving the electron, Cu A reduces the 6-coordinate, low-spin, bis-Hi...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

2006

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Designing Light‐Activated Charge‐Separating Proteins with a Naphthoquinone Amino Acid

et al. 2015

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The first principles design of manmade redox-protein maquettes is used to clarify the physical/ chemical engineering supporting the mechanisms of natural enzymes with a view to recapitulate and surpass natural performance. Herein, we use intein-based protein semisynthesis to pair a synthetic naphthoquinone amino acid (Naq) with histidineligated photoactive metal-tetrapyrrole cofactors, creating a 100 μs photochemical charge separation unit akin to photosynthetic reaction centers. By using propargyl groups to protect the redox-active para-quinone during synthesis and assembly while permitting selective activation, we gain the ability to employ the quinone amino acid redox cofactor with the full set of natural amino acids in protein design. Direct anchoring of quinone to the protein backbone permits secure and adaptable control of intraprotein electrontunneling distances and rates.Correspondence to: Christopher C. Moser, moserc@mail.med.upenn.edu.[+] These authors contributed equally to this work. Present address: Max Planck Institute for Developmental Biology T4bingen, 72076 (Germany) Supporting information (including the Experimental Section) and ORCID(s) from the author(s) for this article are available on the WWW under http://dx.doi.org/10.1002/anie.201507094. Protein semisynthesis is a powerful method for unraveling the physical/chemical engineering of natural enzyme mechanisms through the introduction of post-translational modifications, biophysical probes, or unnatural amino acids into proteins of any size. [1][2][3][4] As one example, semisynthetic intein chemistry was used to insert fluorinated tyrosines with altered phenolic pK a values, but not altered redox potentials, to resolve the role of proton transfer in intraprotein radical electron transfer catalysis in ribonucleotide reductase. [5] Some of the most fundamental questions of natural-redox-protein engineering involve the physical/chemical interplay between redox cofactors and their protein environment. [6][7][8][9] Site-directed mutagenesis around cofactors and reconstitution with exotic cofactors are also important methods to clarify electron-transfer engineering. [10][11][12][13] However, as products of unplanned natural selection, native protein sequences tend to accumulate obscure and often unresolvable interdependencies between amino acids themselves and between amino acids and cofactors. As an alternative, we construct a wide range of artificial redox proteins that have minimal reference to any specific natural protein sequence and instead include simplified, first-principles-directed protein folding and reduced amino acid diversity. [14][15][16] These provide a platform to clarify the typical properties of redox cofactors in an unselected protein environment, identify what elements of protein engineering are effective in controlling the activity of redox cofactors in proteins, and resolve the roles of individual amino acids. HHS Public AccessOur work on securing redox-active tetrapyrrole cofactors, such as hemes, chlorins, and b...

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Designing Light‐Activated Charge‐Separating Proteins with a Naphthoquinone Amino Acid

et al. 2015

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Electrostatic Environment of Hemes in Proteins: pK_as of Hydroxyl Ligands

Cited by 30 publications

References 86 publications

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

Designing Light‐Activated Charge‐Separating Proteins with a Naphthoquinone Amino Acid

Designing Light‐Activated Charge‐Separating Proteins with a Naphthoquinone Amino Acid

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Electrostatic Environment of Hemes in Proteins: pKas of Hydroxyl Ligands

Cited by 30 publications

References 86 publications

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

Designing Light‐Activated Charge‐Separating Proteins with a Naphthoquinone Amino Acid

Designing Light‐Activated Charge‐Separating Proteins with a Naphthoquinone Amino Acid

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Electrostatic Environment of Hemes in Proteins: pK_as of Hydroxyl Ligands