Heme−Peptide Models for Hemoproteins. 1. Solution Chemistry of <i>N</i>-Acetylmicroperoxidase-8

Munro, Orde Q.; Marques, Helder M.

doi:10.1021/ic9502842

Cited by 93 publications

(146 citation statements)

References 127 publications

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“…The b-type waterhemes have an Em,sol of -120 mV, while the a-type heme in cytochrome c oxidase has an Em,sol of -20 mV. Hydroxyl-hemes have Em,sols 80 mV lower than the water-hemes (72,73), consistent with the 1.3 pKa unit shift between the pKas of ferric and ferrous aquo-heme (70,71). b The pKa and Em of the aquo-heme a3 is calculated with CuA oxidized and heme a and CuB reduced to mimic the experimental pKa measurements in the CO photolyzed mixed-valence complex (53).…”

Section: Resultsmentioning

confidence: 62%

Electrostatic Environment of Hemes in Proteins: pK_as of Hydroxyl Ligands

2006

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The pK a s of ferric aquo-heme and aquo-heme electrochemical midpoints (E m s) at pH 7 in sperm whale myoglobin, Aplysia myoblogin, hemoglobin I, heme oxygenase 1, horseradish peroxidase and cytochrome c oxidase were calculated with Multi-Conformation Continuum Electrostatics (MCCE). The pK a s span 3.3 pH units from 7.6 in heme oxygenase 1 to 10.9 in peroxidase, and the E m s range from -250 mV in peroxidase to 125 mV in Aplysia myoglobin. Proteins with higher in situ ferric aquo-heme pK a s tend to have lower E m s. Both changes arise from the protein stabilizing a positively charged heme. However, compared with values in solution, the protein shifts the aquo-heme E m s more than the pK a s. Thus, the protein has a larger effective dielectric constant for the protonation reaction, showing that electron and proton transfers are coupled to different conformational changes that are captured in the MCCE analysis. The calculations reveal a breakdown in the classical continuum electrostatic analysis of pairwise interactions. Comparisons with DFT calculations show that Coulomb's law overestimates the large unfavorable interactions between the ferric water-heme and positively charged groups facing the heme plane by as much as 60%. If interactions with Cu B in cytochrome c oxidase and Arg 38 in horseradish peroxidase are not corrected, the pK a calculations are in error by as much as 6 pH units. With DFT corrected interactions calculated pK a s and E m s differ from measured values by less than 1 pH unit or 35 mV, respectively. The in situ aquo-heme pK a is important for the function of cytochrome c oxidase since it helps to control the stoichiometry of proton uptake coupled to electron transfer [Song, Michonova-Alexova, and Gunner ( . Heme oxygenase is an essential protein in heme metabolism, using three O 2 s and seven electrons to degrade heme to biliverdin (3). Peroxidases reduce toxic hydrogen peroxide to water and then carry out one electron oxidization of a wide range of substrates (4,5). Cytochrome c oxidase is the terminal electron acceptor in the respiratory chain (6-9). Here Heme a 3 forms a binuclear center with a copper complex (Cu B ) that reduces O 2 to water. The protein uses the released chemical energy to pump protons across the membrane. Other five-coordinate heme proteins carry out NO storage and transport (10), steroid synthesis (11, 12), and O 2 , CO, and NO sensing (13,14).Hemes can carry out many biological functions because the protein, especially the active site residues, modulates the ligand binding specificity and resultant chemistry. The diversity of in situ heme functions highlights important interactions of proteins with their cofactors and substrates. Extensive studies have explored how ligand geometry (15-17) and the protein scaffolding (18) affect in situ heme properties (1). The range of redox free energy and pK a s of protein-bound hemes and their ligands show the importance of electrostatic interactions (19,20). For example, sixcoordinate bis-His-hemes have E m s ranging f...

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Section: Resultsmentioning

confidence: 62%

Electrostatic Environment of Hemes in Proteins: pK_as of Hydroxyl Ligands

2006

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“…To couple proton uptake to BNC reduction, the hydroxyl pK a must be lower than the solution pH in the fully oxidized state and higher following reduction. The pK a,sol for deprotonation of an oxidized Hisaquo-heme has been measured to be 9.6 (86), while that of an oxidized Cu(II) with 3 His ligands is estimated to be 9.4 (88,89). In the fully oxidized protein, MCCE Monte Carlo sampling places a hydroxyl on Cu B down to a pH below 4 and a water on heme a 3 beyond pH 11.…”

Section: Resultsmentioning

confidence: 99%

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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“…A different approach involves the use of heme-peptide complexes (called microperoxidases) obtained from the proteolysis of horse heart cytochrome c [20,21]. However, while bearing the advantages of good water solubility and reduced tendency to aggregate, which are helpful for the comparison with the proteins, microperoxidases suffer the problem of containing a different type of heme (heme c) in their structure.…”

Section: Introductionmentioning

confidence: 99%

pH-dependent redox and CO binding properties of chelated protoheme-l-histidine and protoheme-glycyl-l-histidine complexes

et al. 2005

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The pH dependence of redox properties, spectroscopic features and CO binding kinetics for the chelated protohemin-6(7)-L-histidine methyl ester (heme-H) and the chelated protohemin-6(7)-glycyl-L-histidine methyl ester (heme-GH) systems has been investigated between pH 2.0 and 12.0. The two heme systems appear to be modulated by four protonating groups, tentatively identified as coordinated H 2 O, one of heme's propionates, Ne of the coordinating imidazole, and the carboxylate of the histidine residue upon hydrolysis of the methyl ester group (in acid medium). The pK a values are different for the two hemes, thus reflecting structural differences. In particular, the different strain at the Fe-N e bond, related to the different length of the coordinating arm, results in a dramatic alteration of the bond strength, which is much smaller in heme-H than in heme-GH. It leads to a variation in the variation of the pKa for the protonation of the N e of the axial imidazole as well as in the proton-linked behavior of the other protonating groups, envisaging a cross-talk communication mechanism among different groups of the heme, which can be operative and relevant also in the presence of the protein matrix.

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Heme−Peptide Models for Hemoproteins. 1. Solution Chemistry of N-Acetylmicroperoxidase-8

Cited by 93 publications

References 127 publications

Electrostatic Environment of Hemes in Proteins: pK_as of Hydroxyl Ligands

Electrostatic Environment of Hemes in Proteins: pK_as of Hydroxyl Ligands

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

pH-dependent redox and CO binding properties of chelated protoheme-l-histidine and protoheme-glycyl-l-histidine complexes

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Heme−Peptide Models for Hemoproteins. 1. Solution Chemistry of N-Acetylmicroperoxidase-8

Cited by 93 publications

References 127 publications

Electrostatic Environment of Hemes in Proteins: pKas of Hydroxyl Ligands

Electrostatic Environment of Hemes in Proteins: pKas of Hydroxyl Ligands

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

pH-dependent redox and CO binding properties of chelated protoheme-l-histidine and protoheme-glycyl-l-histidine complexes

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Product

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Electrostatic Environment of Hemes in Proteins: pK_as of Hydroxyl Ligands

Electrostatic Environment of Hemes in Proteins: pK_as of Hydroxyl Ligands