Trans-substitution of the proximal hydrogen bond in myoglobin: I. Structural consequences of hydrogen bond deletion

Barrick, Doug; Dahlquist, Frederick W.

doi:10.1002/(sici)1097-0134(20000601)39:4<278::aid-prot20>3.0.co;2-t

Cited by 18 publications

(32 citation statements)

References 37 publications

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“…The X-ray crystal structure of the metaquo form of H93G(4-MeIm) and H93G(1-MeIm) show nearly identical conformations for methyl imidazole in the proximal pocket [34]. The X-ray structure for H93G(4-MeIm) is consistent with hydrogen bonding for 4-methyl imidazole with S92 that is stronger than in wild-type Mb.…”

Section: H93g(1-meim) Probes Hydrogen Bonding In the Proximal Pocketmentioning

confidence: 72%

Proximal ligand motions in H93G myoglobin

Franzen

Peterson

Brown

et al. 2002

European Journal of Biochemistry

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Resonance Raman spectroscopy has been used to observe changes in the iron-ligand stretching frequency in photoproduct spectra of the proximal cavity mutant of myoglobin H93G. The measurements compare the deoxy ferrous state of the heme iron in H93G(L), where L is an exogenous imidazole ligand bound in the proximal cavity, to the photolyzed intermediate of H93G(L)*CO at 8 ns. There are significant differences in the frequencies of the iron-ligand axial out-of-plane mode m(Fe-L) in the photoproduct spectra depending on the nature of L for a series of methylsubstituted imidazoles. Further comparison was made with the proximal cavity mutant of myoglobin in the absence of exogenous ligand (H93G) and the photoproduct of the carbonmonoxy adduct of H93G (H93G-*CO). For this case, it has been shown that H 2 O is the axial (fifth) ligand to the heme iron in the deoxy form of H93G. The photoproduct of H93G-*CO is consistent with a transiently bound ligand proposed to be a histidine. The data presented here further substantiate the conclusion that a conformationally driven ligand switch exists in photolyzed H93G-*CO. The results suggest that ligand conformational changes in response to dynamic motions of the globin on the nanosecond and longer time scales are a general feature of the H93G proximal cavity mutant.Keywords: resonance Raman, heme, myoglobin, hemoglobin, ligand switch.Protein structural relaxation following heme photolysis has been studied in globins as a means to obtain information on structural intermediates following diatomic ligand photolysis. In hemoglobin (Hb), time-resolved spectroscopic studies have provided information on the time scale for transition from the six-coordinate R state to the fivecoordinate T-state [1][2][3]. The proximal cavity mutant of Hb has recently demonstrated the key role of the proximal histidine in the cooperativity of quaternary structure change in response to ligand binding [4]. Strain in the covalent bond to the heme iron of Hb can be monitored by following the shift in frequency of the iron-histidine axial mode, m(FeHis), by time-resolved resonance Raman spectroscopy [5]. In myoglobin (Mb), these studies have indicated a much smaller change in structure [6]: the observed frequency shift of the iron-histidine band is c. 1.6 cm )1 on the 8 ns time scale compared to 12 cm )1 in Hb. Nonetheless, this shift in the m(Fe-His) Raman band is significant because shifts in absorption bands (the time-dependent Soret band shift and band III shift) have been attributed to iron out-of-plane displacement that should also be coupled to m(Fe-His) [7][8][9][10]. A structural interpretation of these observable phenomena helps to bridge the gap between the extensive X-ray crystallography studies and the thermodynamic and kinetic data available for Mb [7,[11][12][13][14][15][16][17][18][19][20].Histidine-ligated heme enzymes have a surprisingly large range of functions. In peroxidase, a charge relay due to hydrogen bonding of the imidazole ring of histidine permits the formation of high valent iro...

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Section: H93g(1-meim) Probes Hydrogen Bonding In the Proximal Pocketmentioning

confidence: 72%

Proximal ligand motions in H93G myoglobin

Franzen

Peterson

Brown

et al. 2002

European Journal of Biochemistry

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“…The shift from R to T state hemoglobin causes His F8 to pull the iron atom slightly out of the plane of the heme resulting in an approximate 800‐fold reduction in oxygen affinity 14, 15. The degree to which the proximal His F8 affects reactivity and protein stability in Mb and human hemoglobin has been a recent focus of research 16–18…”

Section: Introductionmentioning

confidence: 99%

The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme

et al. 2002

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Sperm whale myoglobin (Mb) and soybean leghemoglobin (Lba) are two small, monomeric hemoglobins that share a common globin fold but differ widely in many other aspects. Lba has a much higher affinity for most ligands, and the two proteins use different distal and proximal heme pocket regulatory mechanisms to control ligand binding. Removal of the constraint provided by covalent attachment of the proximal histidine to the F-helices of these proteins decreases oxygen affinity in Lba and increases oxygen affinity in Mb, mainly because of changes in oxygen dissociation rate constants. Hence, Mb and Lba use covalent constraints in opposite ways to regulate ligand binding. Swapping the F-helices of the two proteins brings about similar effects, highlighting the importance of this helix in proximal heme pocket regulation of ligand binding. The F7 residue in Mb is capable of weaving a hydrogen-bonding network that holds the proximal histidine in a fixed orientation. On the contrary, the F7 residue in Lba lacks this property and allows the proximal histidine to assume a conformation favorable for higher ligand binding affinity. Geminate recombination studies indicate that heme iron reactivity on picosecond timescales is not the dominant cause for the effects observed in each mutation. Results also indicate that in Lba the proximal and distal pocket mutations probably influence ligand binding independently. These results are discussed in the context of current hypotheses for proximal heme pocket structure and function.

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“…Typically, 150 of these 10-ml cultures were combined for protein purification. Purification was as described (19), except that ammonium sulfate precipitation was omitted. Instead, cleared lysates were dialyzed overnight against 10 mM imidazole hydrochloride (pH 6.5) before passage over sequential DE52 and CM52 ion-exchange columns.…”

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confidence: 99%

Nonspecific hydrophobic interactions stabilize an equilibrium intermediate of apomyoglobin at a key position within the AGH region

Bertagna

Barrick

2004

Proc. Natl. Acad. Sci. U.S.A.

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Acid-induced unfolding of apomyoglobin (apoMb) proceeds in a multistate process involving at least one equilibrium intermediate (I) at pH 4.2. The structure of the I form has been investigated thoroughly, with significant effort devoted to identifying potentially stabilizing native contacts. Here, we test whether rigid side-chain packing interactions like those in holomyoglobin persist at a buried position, Met-131, within the low-pH apoMb intermediate. We have measured the urea-induced unfolding transitions of overpacking, underpacking, and polar substitutions of Met-131 to determine the effect on the stability of the native and intermediate states of apoMb. Whereas underpacking substitutions should destabilize the I form irrespective of the degree of native side-chainpacking interactions, we anticipate that overpacking replacements might show opposite effects in a tightly packed environment, compared with a region lacking native side-chain packing interactions. We observe that, whereas underpacking and polar substitutions destabilize the I form, overpacking substitutions are stabilizing, implying that I is structurally plastic. We also report a strong correlation between the I state unfolding free energies and side-chain transfer free energies from water to octanol. Our results suggest that, whereas side-chain hydrophobicity is important for the stability of the I form, specific side-chain packing interactions are not.

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Trans-substitution of the proximal hydrogen bond in myoglobin: I. Structural consequences of hydrogen bond deletion

Cited by 18 publications

References 37 publications

Proximal ligand motions in H93G myoglobin

Proximal ligand motions in H93G myoglobin

The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme

Nonspecific hydrophobic interactions stabilize an equilibrium intermediate of apomyoglobin at a key position within the AGH region

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