Replacement of the Proximal Ligand of Sperm Whale Myoglobin with Free Imidazole in the Mutant His-93.fwdarw.Gly

Barrick, Doug

doi:10.1021/bi00187a023

Cited by 185 publications

(258 citation statements)

References 29 publications

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“…This results in a small downfield shift in the resonances of the R and γ meso protons, as seen in H93G(Im)CO compared to WT MbCO. Thus, the chemical shift differences of the R and γ meso protons between WT MbCO and H93G(Im)CO are consistent with the rotation of the Im ring plane relative to H93 seen in the X-ray structure of the metaquo form (Barrick, 1994) and the NMR spectrum of the metcyano complex (Decatur & Boxer, 1995a).…”

Section: Structural Features Of H93g(l)cosupporting

confidence: 78%

“…CO recombination rates were measured by flash photolysis as described elsewhere (Lambright et al, 1989(Lambright et al, , 1994. For the complexes presented here, the final phase of CO recombination is the only phase which FIGURE 1: (A) Schematic diagram of the heme pocket in H93G-(Im)CO, assuming it is the same as found for the metaquo form (Barrick, 1994). The proximal imidazole can be exchanged with many exogenous ligands (L) from solution (DePillis et al, 1994).…”

Section: Methodsmentioning

confidence: 99%

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Modulation of Protein Function by Exogenous Ligands in Protein Cavities: CO Binding to a Myoglobin Cavity Mutant Containing Unnatural Proximal Ligands

1996

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A variety of heterocyclic ligands can be exchanged into the proximal cavity of sperm whale myoglobin mutant H93G, providing a simple method for introduction of the equivalent of unnatural amino acid side chains into a functionally critical location in this protein. These modified proteins bind CO on the distal side. 1 H NMR data on H93G(Im)CO, where Im is imidazole, demonstrate that the structure of the distal heme pocket in H93G(Im)CO is very similar to that of wild type; thus, the effects of the proximal ligand's properties on CO binding can be studied with minimal perturbation of distal pocket structure. The exogenous proximal ligands used in this study include imidazole (Im), 4-methylimidazole (4-MeIm), 4-bromoimidazole (4-BrIm), N-methylimidazole (N-MeIm), pyridine (Pyr), and 3-fluoropyridine (3-FPyr). Substitution of the proximal ligand is found to produce substantial changes in the CO on and off rates, the equilibrium binding constant, and the vibrational stretch frequency of CO. Many of the changes are as large as those reported for distal pocket mutants prepared by site-directed mutagenesis. The ability to systematically vary the nature of the proximal ligand is exploited to test the effects of particular properties of the proximal ligand on CO binding. For example, 4-MeIm and 4-BrIm are similar in size and shape but differ significantly in pK a . The same relationship is true for Pyr and 3-FPyr. By comparison of the IR spectra and CO recombination kinetics of these complexes, the effects of proximal ligand pK a on the CO binding are assessed. Likewise, N-MeIm and 4-MeIm are similar in size and pK a but differ in their ability to hydrogen bond to amino acid residues in the proximal cavity. Comparisons of IR spectra and CO binding kinetics in these complexes reveal that proximal ligand conformation and hydrogen bonding affect the kinetics of CO binding. The mechanism of proximal ligand exchange between solution and the proximal cavity in CO complexes was investigated by obtaining the 19 F NMR spectrum of H93G(3-FPyr)CO, whose 19 F signal can be observed without interference from resonances of the protein. The proximal ligand is found to exchange within a few seconds by saturation transfer. This exchange rate is about 2 orders of magnitude faster than what is observed for the isoelectronic metcyano complex [Decatur, S. M., & Boxer, S. G. (1995) Biochemistry 34, 2122-2129; in both the ferrous CO and ferric cyano complexes, the proximal ligand exchange rate is independent of ligand concentration. These results suggest that the rate-limiting step in proximal ligand exchange is breakage of the iron-ligand bond, followed by rapid diffusion of the ligand through the protein to bulk solution.

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Section: Structural Features Of H93g(l)cosupporting

confidence: 78%

Section: Methodsmentioning

confidence: 99%

Modulation of Protein Function by Exogenous Ligands in Protein Cavities: CO Binding to a Myoglobin Cavity Mutant Containing Unnatural Proximal Ligands

1996

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confidence: 94%

“…A successful example of this is provided by the sperm whale myoglobin (Mb) H93G mutant, which has been extensively used for this purpose. 8,9 The crystal structures of β-mercaptoethanol and acetate-bound Mb H93G have recently confirmed that the exogenous ligands bind in the proximal ligation site. 10 In this report, we report an initial evaluation of the coordination of selenolate ligands to the heme in both CYP101 and the heme oxygenase-1 H25A cavity mutant.…”

mentioning

confidence: 94%

Selenolate Complexes of CYP101 and the Heme-Bound hHO-1/H25A Proximal Cavity Mutant

Jiang

Montellano

2008

Inorg. Chem.

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Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-visible spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions, but reduced the heme iron to the ferrous state as shown by formation of a ferrous-CO complex. Under aerobic conditions, the selenolate but not thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect.Cytochrome P450 enzymes are a superfamily of heme proteins that employ a cysteine thiolate as the proximal ligand to the heme. 1 The P450-catalyzed reaction has been thought for three decades to be mediated by a Compound I-like ferryl species that is generated by reductive activation of molecular oxygen at the prosthetic heme iron center. Despite support by a variety of data for a Compund I-like ferryl species in the P450 catalytic cycle, it has remained elusive towards spectroscopic detection. 2,3 Conflicting results have also been obtained from determinations of the lifetime of the ferryl species using radical clock substrates. 4 Direct detection and characterization of the reactive oxidizing species in P450 enzymes would therefore be highly desirable. Recent computational studies have suggested that a seleno-CYP101 mutant in which the cysteine ligand is replaced by selenocysteine might provide an approach to investigation of the elusive P450 oxidizing species. 5 The Se-Compound I intermediate was predicted to form faster than the wild-type S-Compound I, and to be consumed more slowly. The increased lifetime of the intermediate suggested by the calculations indicated that it might become spectroscopically detectable.Targeted insertion of selenocysteine into a protein sequence through genetic code manipulations is a complex cotranslational process not easily adapted to P450 enzymes. 6,7 An alternative and straightforward approach to investigate the effect of ligands in heme proteins has been to use a cavity mutant in which the proximal amino acid ligand is replaced by a small and noncoordinating amino acid such as an alanine or glycine, creating a pocket that can accommodate exogenous unnatural ligands. A successful example of this is provided by the sperm whale myoglobin (Mb) H93G mutant, which has been extensively used for this purpose. 8,9 The crystal structures of β-mercaptoethanol and acetate-bound Mb H93G have recently confirmed that the exogenous ligands bind in the proximal ligation site.

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“…The heme iron in Mb is coordinated to His 93 in all Mbs. When this residue is replaced by glycine and the mutant protein H93G is expressed in bacteria grown in the presence of exogenous imidazole (Im), a stable protein is formed containing Im in the cavity created by the mutation (Barrick, 1994). Im serves as the axial ligand to heme in this protein denoted H93G- (Im).…”

mentioning

confidence: 99%

Mg Coordination by Amino Acid Side Chains Is Not Required for Assembly and Function of the Special Pair in Bacterial Photosynthetic Reaction Centers

1996

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A conserved histidine serves as the axial ligand to the Mg of bacteriochlorophylls in the photosynthetic reaction center (RC) and many other photosynthetic systems. The histidine axial ligands to each and both bacteriochlorophylls of the special-pair primary electron donor of the Rhodobacter sphaeroides RC have been replaced with glycine to create a cavity. In each case, RCs assemble and a normal special-pair comprised of Mg-containing bacteriochlorophylls is formed, as judged by many different spectroscopic and functional probes (e.g., absorption and Stark spectra, *P decay kinetics, P + Q Arecombination rate, and the redox potential of P). In contrast with heme proteins, where this strategy has been exploited to introduce exogenous organic ligands that can greatly affect the functional properties of the protein [DePillis, G. D., Decatur, S. M., Barrick, D., & Boxer, S. G. (1994) J. Am. Chem. Soc. 116,[6981][6982], addition of exogenous imidazole, pyridine, and ethanethiol has no measurable effect on the functional properties of the special pair in these cavity mutants. FT-Raman spectroscopy is used to provide more detailed information on local interactions around the special pair. Data in the core-size marker mode and carbonyl stretching region suggest that an adventitious ligand replaces histidine as the axial ligand to bacteriochlorophylls in the cavity mutants. We speculate that this ligand is water. Furthermore, the position of the core-size marker mode changes when the cavity mutant RCs are incubated with exogenous ligands such as imidazole, pyridine, or ethanethiol, suggesting that the axial ligand to the special pair BChls can be exchanged in the cavity mutants. Interestingly the temperature dependence of P + Q A -recombination kinetics is very similar in the cavity mutants and WT, suggesting that the axial ligands to the special pair are not significant contributors to the solvent reorganization energy for this reaction. These results lead to the surprising conclusion that the nature of the axial ligand to the special pair has little influence on the properties of the macrocycle, and that axial coordination from the protein by histidine is not required for bacteriochlorophyll binding or for efficient electron transfer in the RC.

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Replacement of the Proximal Ligand of Sperm Whale Myoglobin with Free Imidazole in the Mutant His-93.fwdarw.Gly

Cited by 185 publications

References 29 publications

Modulation of Protein Function by Exogenous Ligands in Protein Cavities: CO Binding to a Myoglobin Cavity Mutant Containing Unnatural Proximal Ligands

Modulation of Protein Function by Exogenous Ligands in Protein Cavities: CO Binding to a Myoglobin Cavity Mutant Containing Unnatural Proximal Ligands

Selenolate Complexes of CYP101 and the Heme-Bound hHO-1/H25A Proximal Cavity Mutant

Mg Coordination by Amino Acid Side Chains Is Not Required for Assembly and Function of the Special Pair in Bacterial Photosynthetic Reaction Centers

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