Ligand binding to heme proteins: connection between dynamics and function

Kriegl

Journal of Biological Chemistry

2004

132

We have examined the effects of active site residues on ligand binding to the heme iron of mouse neuroglobin using steady-state and time-resolved visible spectroscopy. Absorption spectra of the native protein, mutants H64L and K67L and double mutant H64L/K67L were recorded for the ferric and ferrous states over a wide pH range (pH 4 -11), which allowed us to identify a number of different species with different ligands at the sixth coordination, to characterize their spectroscopic properties, and to determine the pK values of active site residues. In flash photolysis experiments on CO-ligated samples, reaction intermediates and the competition of ligands for the sixth coordination were studied. These data provide insights into structural changes in the active site and the role of the key residues His 64 and Lys 67 . His 64 interferes with exogenous ligand access to the heme iron. Lys 67 sequesters the distal pocket from the solvent. The heme iron is very reactive, as inferred from the fast ligand binding kinetics and the ability to bind water or hydroxyl ligands to the ferrous heme. Fast bond formation favors geminate rebinding; yet the large fraction of bimolecular rebinding observed in the kinetics implies that ligand escape from the distal pocket is highly efficient. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH, which may be important in functional processes.

Section: Resultsmentioning

confidence: 92%

Structural Dynamics in the Active Site of Murine Neuroglobin and Its Effects on Ligand Binding

Kriegl

Journal of Biological Chemistry

2004

132

“…2 A) shows strong rebinding already at 3 K, implying very low activation-energy barriers for geminate rebinding from the heme pocket. A pronounced kinetic hole burning shift (24,25) with temperature in A 1 , ranging from 1,937 to 1,947 cm Ϫ1 , reflects substantial heterogeneity of the CO environment in this bound-state conformation.…”

Section: Experimental Results and Interpretationmentioning

confidence: 98%

“…Hence, the entry process controls the apparent bimolecular rate, 1 Ϸ k SB (25,31). At high temperature, however, the inner barrier between states B and A governs the kinetics, 1 Ϸ k BA ϫ P B , with the (weakly temperature-dependent) pocket occupation factor P B ϭ (k SB ͞k BS ).…”

Section: [1]mentioning

confidence: 99%

Ligand binding and protein dynamics in neuroglobin

Kriegl

Bhattacharyya

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

et al. 2002

144

210

Neuroglobin (Ngb) is a recently discovered protein in vertebrate brain tissue that belongs to the globin family of proteins. It has been implicated in the neuronal response to hypoxia or ischemia, although its physiological role has been hitherto unknown. Ngb is hexacoordinate in the ferrous deoxy form under physiological conditions. To bind exogenous ligands like O2 and CO, the His E7 endogenous ligand is displaced from the sixth coordination. By using infrared spectroscopy and nanosecond time-resolved visible spectroscopy, we have investigated the ligand-binding reaction over a wide temperature range (3-353 K). Multiple, intrinsically heterogeneous distal heme pocket conformations exist in NgbCO. Photolysis at cryogenic temperatures creates a five-coordinate deoxy species with very low geminate-rebinding barriers. The photodissociated CO is observed to migrate within the distal heme pocket even at 20 K. Flash photolysis near physiological temperature (275-353 K) exhibits four sequential kinetic features: (i) geminate rebinding (t < 1 s); (ii) extremely fast bimolecular exogenous ligand binding (10 s < t < 1 ms) with a nontrivial temperature dependence; (iii) endogenous ligand binding (100 s < t < 10 ms), which can be studied by using flash photolysis on deoxy Ngb; and (iv) displacement of the endogenous by the exogenous ligand (10 ms < t < 10 ks). All four processes are markedly nonexponential, suggesting that Ngb fluctuates among different conformations on surprisingly long time scales.G lobins are proteins that bind dioxygen and other small ligands at the central iron of a heme prosthetic group embedded in a highly conserved ␣-helical ''globin'' fold. Hemoglobin (Hb) and myoglobin (Mb) are the most prominent members of this protein family (1). Interrelations among structure, dynamics, and function in globins have been investigated in great detail, most likely more thoroughly than for any other protein family. Mb especially has, for a long time, served as a paradigm in the biological physics of proteins (2, 3).Recently, two new members have joined the globin family, cytoglobin (4) and neuroglobin (Ngb) (5). The latter consists of a single chain of 151 aa. Ngb, which occurs in neurons, has less than 25% sequence identity with Mb or Hb but nevertheless displays all key determinants of the globin fold (6). It has moderate oxygen affinity and seems most closely related to the globin found in the glial cells of the annelid Aphrodite (7). The discovery of a six-coordinate heme in the deoxy form of Ngb (5), with the His E7 side chain occupying the sixth coordination, came somewhat as a surprise because it was believed for a long time that a pentacoordinate heme iron, with a vacant ligandbinding site, was a common characteristic of globins. In recent years, however, hexacoordinate globins also have been isolated from bacteria, unicellular eukaryotes, and plants [nonsymbiotic Hbs, nsHbs (8,9), and truncated Hbs, trHbs (10)]. In these proteins, the exogenous ligand has to compete with an intramolecular side chain for ...

European Journal of Biochemistry

“…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 iron oxidation states that play a role in the redox function of these enzymes [21][22][23].…”

mentioning

confidence: 99%

Proximal ligand motions in H93G myoglobin

Franzen

Peterson

Brown

et al. 2002

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...