Proximal and Distal Influences on Ligand Binding Kinetics in Microperoxidase and Heme Model Compounds

Cao, Wei; X, Ye; Gy, Georgiev; Berezhna, Svitlana; Sjodin, Theodore; Aa, Demidov; Wang, W; Jt, Sage; Pm, Champion

doi:10.1021/bi0497291

Cited by 33 publications

(50 citation statements)

References 75 publications

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“…These comparisons are consistent with the general idea that structural flexibility is more advantageous for functional ligand-binding heme proteins than for functional electron-transfer proteins, as indicated in the Introduction. Apart from our finding of variation of the CO rebinding kinetics in differently engineered proteins, it is clear that the protein matrix must be responsible for the fast rebinding, as geminate rebinding to the 5-coordinate heme iron in microperoxidase does not occur at low concentrations (25) (it does in aggregates (25,58)). …”

Section: Discussionmentioning

confidence: 84%

“…In a similar way, in wild type and mutant Mb, multiphasic picosecond heme-NO recombination kinetics have proven a sensitive tool for assessing the dynamic, steric, and electrostatic role of the heme environment (16 -22). As mentioned above, in Mb, CO geminate recombination is usually less extensive and takes place on a much longer time scale (23)(24)(25) and in the native protein in a monophasic way ( ϭ ϳ150 ns). To gain insight into the dynamic properties of the hydrophobic heme-binding core of cyt.…”

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confidence: 96%

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Ligand Dynamics in an Electron Transfer Protein

Silkstone

Jasaitis

Wilson

et al. 2007

Journal of Biological Chemistry

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Substitution of the heme coordination residue Met-80 of the electron transport protein yeast iso-1-cytochrome c allows external ligands like CO to bind and thus increase the effective redox potential. This mutation, in principle, turns the protein into a quasi-native photoactivable electron donor. We have studied the kinetic and spectral characteristics of geminate recombination of heme and CO in a series of single M80X (X ‫؍‬ Ala, Ser, Asp, Arg) mutants, using femtosecond transient absorption spectroscopy. In these proteins, all geminate recombination occurs on the picosecond and early nanosecond time scale, in a multiphasic manner, in which heme relaxation takes place on the same time scale. The extent of geminate recombination varies from >99% (Ala, Ser) to ϳ70% (Arg), the latter value being in principle low enough for electron injection studies. The rates and extent of the CO geminate recombination phases are much higher than in functional ligand-binding proteins like myoglobin, presumably reflecting the rigid and hydrophobic properties of the heme environment, which are optimized for electron transfer. Thus, the dynamics of CO recombination in cytochrome c are a tool for studying the heme pocket, in a similar way as NO in myoglobin. We discuss the differences in the CO kinetics between the mutants in terms of the properties of the heme environment and strategies to enhance the CO escape yield. Experiments on double mutants in which Phe-82 is replaced by Asp or Gly as well as the M80D substitution indicate that such steric changes substantially increase the motional freedom-dissociated CO.

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Section: Discussionmentioning

confidence: 84%

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confidence: 96%

Ligand Dynamics in an Electron Transfer Protein

Silkstone

Jasaitis

Wilson

et al. 2007

Journal of Biological Chemistry

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“…The dynamics associated with ligand binding to the heme group has been studied for many years in both native and mutant proteins [3][4][5][6][7][8][9][10][11][12][13][14][15] and in heme model compounds. [16][17][18][19][20][21][22][23][24] The binding process for model compounds involves at least two steps: the diffusion of ligand through the surrounding solvent or buffer followed by the bond formation step between the diatomic ligand and the iron. In proteins, the process is complicated by the protein matrix where various entry and exit channels, as well as docking sites and cavities within the protein, lead to a complexity in the overall reaction process that is still not fully understood.…”

Section: Introductionmentioning

confidence: 99%

“…40,41 Model compound kinetic studies have been performed in CTAB under conditions where an imidazole ligand is bound to the heme. 18,23 However, for such compounds, the CO ligand escape rate is much larger than its geminate rebinding rate, so the latter process is difficult or impossible to detect. 23 Moreover, in the absence of an added imidazole ligand, the CTAB encapsulated heme samples again display complex kinetic evolution, probably due to a base elimination mechanism 18,42 (i.e., upon CO photolysis, there is a delayed dissociation of the weak proximal base before CO rebinds).…”

Section: Introductionmentioning

confidence: 99%

CO Rebinding to Protoheme: Investigations of the Proximal and Distal Contributions to the Geminate Rebinding Barrier

et al. 2005

J. Am. Chem. Soc.

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The rebinding kinetics of CO to protoheme (FePPIX) in the presence and absence of a proximal imidazole ligand reveals the magnitude of the rebinding barrier associated with proximal histidine ligation. The ligation states of the heme under different solvent conditions are also investigated using both equilibrium and transient spectroscopy. In the absence of imidazole, a weak ligand (probably water) is bound on the proximal side of the FePPIX-CO adduct. When the heme is encapsulated in micelles of cetyltrimethylammonium bromide (CTAB), photolysis of FePPIX-CO induces a complicated set of proximal ligation changes. In contrast, the use of glycerol-water solutions leads to a simple two-state geminate kinetic response with rapid (10-100 ps) CO recombination and a geminate amplitude that can be controlled by adjusting the solvent viscosity. By comparing the rate of CO rebinding to protoheme in glycerol solution with and without a bound proximal imidazole ligand, we find the enthalpic contribution to the proximal rebinding barrier, H p , to be 11 ± 2 kJ/mol. Further comparison of the CO rebinding rate of the imidazole bound protoheme with the analogous rate in myoglobin (Mb) leads to a determination of the difference in their distal free energy barriers: ΔG D ≈ 12 ± 1 kJ/mol. Estimates of the entropic contributions, due to the ligand accessible volumes in the distal pocket and the xenon-4 cavity of myoglobin (~3 kJ/mol), then lead to a distal pocket enthalpic barrier of H D ≈ 9 ± 2 kJ/mol. These results agree well with the predictions of a simple model and with previous independent room-temperature measurements (Tian et al. Phys. Rev. Lett. 1992, 68, 408) of the enthalpic MbCO rebinding barrier (18 ± 2 kJ/mol).

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“…The bound ligand can be further stabilized by electrostatic interactions with surrounding polar amino acid side chains. This E7 gate pathway appears to occur in most if not all animal hemoglobins, with the classic 3-on-3 ␣-helical globin fold and a distal histidine, although experimental verification has only been done rigorously for vertebrate Mbs using time-resolved crystallography (4 -8), site-directed mutagenesis (3,9,11), and time-resolved absorbance and FTIR spectroscopy (12)(13)(14)(15)(16)(17)(18).…”

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confidence: 99%

The Apolar Channel in Cerebratulus lacteus Hemoglobin Is the Route for O2 Entry and Exit

Salter

Nienhaus

et al. 2008

Journal of Biological Chemistry

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The major pathway for O 2 binding to mammalian myoglobins (Mb) and hemoglobins (Hb) involves transient upward movement of the distal histidine (His-64(E7)), allowing ligand capture in the distal pocket. The mini-globin from Cerebratulus lacteus (CerHb) appears to have an alternative pathway between the E and H helices that is made accessible by loss of the N-terminal A helix. To test this pathway, we examined the effects of changing the size of the E7 gate and closing the end of the apolar channel in CerHb by site-directed mutagenesis. Increasing the size of Gln-44(E7) from Ala to Trp causes variation of association (k O2 ) and dissociation (k O2 ) rate coefficients, but the changes are not systematic. More significantly, the fractions (F gem ≈ 0.05-0.19) and rates (k gem ≈ 50 -100 s ؊1) of geminate CO recombination in the Gln-44(E7) mutants are all similar. In contrast, blocking the entrance to the apolar channel by increasing the size of Ala-55(E18) to Phe and Trp causes the following: 1) both k O2 and k O2 to decrease roughly 4-fold; 2) F gem for CO to increase from ϳ0.05 to 0.45; and 3) k gem to decrease from ϳ80 to ϳ9 s ؊1, as ligands become trapped in the channel. Crystal structures and low temperature Fourier-transform infrared spectra of Phe-55 and Trp-55 CerHb confirm that the aromatic side chains block the channel entrance, with little effect on the distal pocket. These results provide unambiguous experimental proof that diatomic ligands can enter and exit a globin through an interior channel in preference to the more direct E7 pathway.The structural mechanisms for O 2 binding to myoglobins (Mb) 3 and hemoglobins (Hb) have been the topic of much research since Kendrew (1) and Perutz et al. (2) first reported the three-dimensional structures of Mb and Hb over 45 years ago. In the case of mammalian myoglobins, ligand binding is well understood (Refs. 3-10 and references therein) and consists of four major steps. Weakly bound water is displaced to create a vacant distal pocket above the heme iron atom. Ligands migrate into the protein through a short channel that is created when the distal histidine (His-64 at the E7 helical position) 4 transiently rotates upward and then are captured in the interior of the distal pocket. This noncovalent intermediate is often called the B state because it can also be generated by photolysis of the equilibrium bound or A state. Covalent bond formation between the internal ligand and the iron atom of the heme group then competes with ligand escape back out through the His(E7) gate. The bound ligand can be further stabilized by electrostatic interactions with surrounding polar amino acid side chains. This E7 gate pathway appears to occur in most if not all animal hemoglobins, with the classic 3-on-3 ␣-helical globin fold and a distal histidine, although experimental verification has only been done rigorously for vertebrate Mbs using time-resolved crystallography (4 -8), site-directed mutagenesis (3, 9, 11), and time-resolved absorbance and FTIR spectroscopy (12-18).In con...

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Proximal and Distal Influences on Ligand Binding Kinetics in Microperoxidase and Heme Model Compounds

Cited by 33 publications

References 75 publications

Ligand Dynamics in an Electron Transfer Protein

Ligand Dynamics in an Electron Transfer Protein

CO Rebinding to Protoheme: Investigations of the Proximal and Distal Contributions to the Geminate Rebinding Barrier

The Apolar Channel in Cerebratulus lacteus Hemoglobin Is the Route for O2 Entry and Exit

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