Hemoglobin, Hb, is the tetrameric oxygen transport protein consisting of two pairs of heme- containing polypeptides, 22, each with tertiary and active site structures generally similar to those of myoglobin. However, owing to the existence of two distinct quaternary structures, labeled T and R, characteristic of the unligated and fully ligated forms, respectively, the affinity of the Hb tetramer for exogenous ligands, including O2, changes dramatically as a function of ligand concentration.25,26Thus, binding of a dioxygen ligand to the lower affinity T-state induces changes in the heme structure—including its conversion from a high-spin ferrous form to a species best formulated as a low-spin ferric superoxide adduct—which consequently lead to changes in the surrounding active site polypeptide structure. The accumulation of forces resulting from these active site structural changes eventually, after two or three sites are ligated, triggers a global
469 δ(CβC1C2) ν7 * ν15 ν7 oop 1463ν28
553 ν25 597 ν24 923 } 1061 ν23 1123 ν22 1252 } 1309 ν21 1393 ν20 1441 1602 ν19
144 ν35 oop
* ν8 751 ν15 781 CH2rock ν6 938 ν32 962 δ(CH3) 1025 ν(C1-C2) ν5 1158 ν30 1219 ν13 1276 CH2 twist 1406 ν29 1481 ν28 1575 ν11 1653 ν10
197 ν34 469 δ(CβC1C2) 1015 ν31 ν22 ν19
144 ν35 226 opp 272 ν9 343 363 674 ν7 806 ν6 1139 ν5 1260 CH2 twist 1318 CH2 wag 1383 ν4 ν29 1519 ν3 ν11 1600 ν2 ν10
ν7 * * oop CH2 wagν21 ν4
* ν(C1-C2) ν5 ν30 ν13 ν4 ν29 }CH2 scissor
δ(CH3) ν11 ν10
}ν8 469 δ(CβC1C2)
* * ν15 oop δ(CH3) ν(C1-C2) ν30 ν13 1464 CH2 scissor
CH2 twist
⊥
⊥
⊥
350 450 550
406.7 530.9 562.8
Wavelength (nm)
B Q0-1 Q0-0
200 400 600 800 1000 1200 1400 1600
Raman shift (cm–1)
Figure 2 Resonance Raman spectra (|| parallel and?perpendicular polarization) of NiOEP. Inset depicts the electronic absorption spectrum of NiOEP (adapted from ref. 12).
Resonance Raman: Bioinorganic Applications 133
structuraltransition, involving specific intra- and inter-subunit contacts, to the R-state quaternary conformation, wherein the remaining unligated sites possess an increased affinity for an exogen- ous ligand. Conversely, dissociation of two or three ligands from the fully ligated tetramer induces a corresponding transition to the lower-affinity T-state structure, from which release of the remaining ligands is facilitated. It is through this cooperative ligand binding process that the O2
transport function of Hb is made so efficient, loading up all four binding sites in a region of high oxygen concentrations—the lungs—while more easily releasing the transported O2 in regions of low concentrations of dioxygen. The interesting analogy has been made of Hb as a molecular lung, ‘‘inhaling’’ dioxygen in the lungs as it undergoes the T–R transition, while ‘‘exhaling’’ its cargo of dioxygen molecules, via the R–T transition, in the oxygen-poor tissues.26As will be seen below, RR and TR3 spectroscopic techniques have proven to be truly exquisite probes of the structure and dynamics of this fascinating allosteric protein.
The heme group structural alterations associated with oxygenation are clearly reflected in the observed RR spectra shown in Figure 3. The oxidation state marker band, 4, shifts from its ferrous-state value of 1,358–1,376 cm1 upon ligation, while shifts of several of the spin-state marker modes, including 3and10, are indicative of a high-spin to low-spin conversion. In the low-frequency region, not shown here, the (Fe–O) stretching mode of the Fe–O2 fragment is clearly identified near 570 cm1, as confirmed by its shift to 548 cm1upon replacement of 16O2
with 18O2.27
Based on the X-ray crystal structures of deoxy and ligated hemoglobins, Perutz formulated a molecular stereochemical mechanism for hemoglobin, a key tenet of which is a strained linkage between the heme iron and the proximalhistidylimidazole bond in the ‘‘tense’’ T-state.28 In an effective demonstration of the power of the technique, RR spectraldata acquired for deoxy Mb, Hb, and chemicalconstructs of hemoglobin that exist in either quaternary state provide direct evidence for the suggested changes in this key linkage. Thus, Kitagawa and co-workers29 employed54/57Fe-labeled hemes to show that the(Fe–Nhis) stretching mode occurs at 223 cm1 in deoxy Mb, while the corresponding modes of deoxy Hb are manifested as a rather asymmetric envelope of bands centered near 216 cm1;Figure 4. Later studies on Mb, employing15N-labeling, confirmed the essential validity of the assignment and helped to further clarify its precise nature.30,31 An important and elegant experiment, first reported by Kitagawa and co-workers32 and later confirmed by others,33employed so-called met-hemoglobin hybrids, (CN)2and (CN)2,
1358 1428 1473 1526 1545 1562 1592 1614
1376 1430 1506 1560 1584 1606 1620 1641
1300 1350 1400 1450 1500 1550 1600 1650 oxy Mb
deoxy Mb
Figure 3 High-frequency resonance Raman spectra of deoxy Mb and oxy Mb.
containing low-spin ferric cyanide adducts in only one type of subunit; the hybrids are converted from an ‘‘R-like’’ quaternary state to one closely resembling the T-state upon the addition of allosteric effectors, such as inositol hexaphosphate, IHP. As shown inFigure 5, the RR spectra of such species, acquired with an excitation line that enhances only the ferrous subunits, document a significant shift to a lower frequency for the(Fe–Nhis) of thesubunits in the T-state, while a much smaller R/T difference is observed for the subunits, implying that the hemes of the subunits of the intact T-state tetramer experience the greater strain in the iron–histidine linkages.
The most attractive approach for characterization of fleeting allosteric intermediates is to exploit the efficient photo-dissociation of CO from the fully ligated adduct, Hb(CO)4, to facilitate transient RR and TR3studies which permit direct interrogation of the structure of these species.
Pioneering studies by Friedman, Rousseau, and others have been concisely summarized in several review articles,34–37with the following examples providing convincing illustrations of the remark- able potential of these methods.
With a sufficiently intense laser pulse, all of the bound CO ligands of the Hb(CO)4precursor are rapidly photolyzed, with the collected scattered light revealing the RR spectrum of the initial photoproduct, a species possessing deligated hemes trapped in an R-state quaternary conform- ation.34While the high-frequency region of the RR spectra provides evidence for small changes in heme macrocycle structure compared with that of equilibrium deoxy Hb,38 more substantial changes are seen in the low-frequency region, including a shift of the (Fe–Nhis) envelope, which now appears as a nearly symmetric band near 228 cm1, as can be seen by inspection of Figure 6; i.e., direct evidence for weakening of the Fe–Nhislinkage in the T-state conformation.34–37 Subsequent (pump/probe) TR3 studies document the temporalevolution of this key(Fe–Nhis) stretching mode through various intermediates, terminating in a frequency quite similar to that of genuine deoxy Hb as the system relaxes (tens of microseconds) to a ‘‘T-like’’ state; the actual terminalstate in this experiment, referred to as T0, is a diligated species, Hb(CO)2, owing to relatively rapid (50 ns) geminate recombination of two CO ligands.39In a recent comprehensive application of these methods, Jayaramanet al. have used visible excitation lines to probe heme prosthetic group structure and ultraviolet lines to monitor the status of key aromatic residues within various intermediates encountered throughout the R to T0 transition.40
216 256 300 340 364 377 403 430 484 542 586
223 239
257 270
303 342 370 405 437 472 499 522 546 562 587
Raman shift (cm–1)
250 300 350 400 450 500 550 deoxy Hb
deoxy Mb
Figure 4 Low-frequency resonance Raman spectra of deoxy Mb and deoxy Hb.
Resonance Raman: Bioinorganic Applications 135
2.11.3.2 Plant Peroxidases
An illustration of the utility of RR for characterizing reactive heme enzymatic intermediates is provided by studies of the plant peroxidases, which contain the same protoheme prosthetic group as Mb, as well as a similar histidine axial ligand, but which present an otherwise different active site environment around the heme, dramatically altering its properties.41,42Thus, in these perox- idases, which catalyze the oxidation of various substrates via heterolytic cleavage of an initial, very short-lived ferric-hydroperoxo intermediate, the proximal histidyl ligand is strongly H-bonded to nearby acceptor groups, rendering a distinct imidazolate character to the coordinated fragment; while the distal pocket contains more hydrophilic residues than that of Mb, including distalhistidine and arginine residues. This active site environment stabilizes the ferric form of the heme in the resting state of the enzyme, and also promotes heterolytic cleavage of the peroxo OO bond; the basic proximal imidazole ligand stabilizes a resulting oxo-iron(IV), ferryl fragment and the positively charged distal arginine helps to stabilize the developing negative charge on the terminal oxo atom. The immediate product of the heterolytic cleavage, called Compound I, which is two oxidation equivalents above the ferric resting state and is usually formulated as a ferrylheme -cation radical, OẳFe(protoporphyrinỵ_), undergoes successive one-electron reductions by oxidation of substrates to first form so-called Compound II, a ferryl heme, OẳFe(protoporphyrin), and then to regenerate the ferric resting state.
Resonance Raman spectroscopy has proven itself as an effective probe in revealing the nature of all of these enzymatic species.19,42,43The increased donor strength of the H-bonded proximal histidylimidazole is evidenced by the observation of a(Fe–Nhis) stretching frequency of 244 cm1
223 302 343 367 406
201 302 343 366 406
223 301 342 366 405 430
219 302 342 367 406
Raman shift (cm–1)
200 250 300 350 400 450
212
a. (αpβpCN)2
b. (αpβpCN)2 + IHP
c. (αpCNβp)2
d. (αpCNβp)2 + IHP
Figure 5 Low frequency resonance Raman spectra of deoxy Hb hybrids (adapted from ref. 33).
in the spectrum of the ferrous form of horseradish peroxidase, HRP, a value significantly higher than that observed for Mb.44 The first intermediate to be studied by RR was the more stable Compound II, which exhibited marker-mode frequencies consistent with the expected low-spin FeIV formulation. In addition, direct evidence was obtained for the presence of an FeẳO fragment from the observation of a band at 776 cm1 which shifted to 745 cm1 when H218
O2
was employed, a shift consistent with that expected for the ferryl fragment.45,46Problems initially encountered in acquiring the spectrum of Compound I, including its efficient photo-degradation in the laser beam,19 were eventually overcome by Palaniappan and Terner, who employed excitation near 350 nm, where the modes of this intermediate are selectively enhanced, relative to those of the photo-product,Figure 7.47The RR spectra of HRP Compound I, later reproduced by other workers,48not only provide direct evidence for the presence of a ferryl fragment (using H218
O2), but also reveal shifts of key marker modes, such as2,3, and4, relative to Compound II, which are consistent with its formulation as a ferryl heme-cation radical. This latter conclusion is supported by studies of modelcompounds, such as Ni(OEPþ_) and others, which exhibit comparable core-mode shifts relative to their neutral parents.19
2.11.3.3 Cytochromes P450
Members of this widely distributed class of heme proteins contain the same protoheme prosthetic group common to the systems described above, but are able to efficiently catalyze some of the most difficult reactions known to occur in biological systems; i.e., the hydroxylation or epoxida- tion of relatively inert hydrocarbon fragments of various substrates, utilizing molecular oxygen as the ultimate source of oxidizing equivalents.49The active site environment in all of these enzymes presents an electron-rich cysteine thiolate axial ligand to the heme and a relatively hydrophobic distalside, possessing only a weak H-bond donor, e.g., a threonine hydroxylgroup. Binding of substrate to the low-spin ferric resting state generates a higher potential high-spin ferric species, triggering a reduction by an associated redox partner. The resulting high-spin ferrous protoheme binds dioxygen to form an adduct best formulated as a ferric-superoxide complex, which is presumably stabilized by a weak H-bonding interaction with the distal side donor. This is the last intermediate sufficiently stable to be definitively characterized by crystallographic or spectro- scopic methods. Delivery of a second electron and a proton apparently facilitates heterolytic cleavage of the putative ferric hydroperoxo species, to generate a remarkably potent hydroxyl- ating intermediate; a reactivity which has earned this species the nickname of ‘‘biological blowtorch.’’50
160 200 240 280 320
222 302
229 302
COHbAm + hν
COHbK+ IHP + hν λexc = 4,200 Å
Raman shift (cm–1)
Figure 6 Low-frequency resonance Raman spectra of the transient deoxy Hb species occurring within 10 ns of photo-dissociating of the carboxyhemoglobin (adapted from ref. 37).
Resonance Raman: Bioinorganic Applications 137
It has been only in the 1990s and early 2000s that high-resolution crystal structures have become available for cytochromes P450 and their substrate-bound analogues.49 Prior to this impressive accomplishment, RR techniques, conducted mainly by Champion and co-workers,51 were quite valuable in providing definitive structural data for various species involved in these systems.19,51Thus, RR studies of the resting state provided direct evidence for its formulation as a low-spin ferriheme, and confirmed its conversion to a high-spin configuration upon binding of substrate, e.g., the 3spin-state marker band shifts to lower frequencies in the substrate-bound form. Evidence for the presence of the electron-rich thiolate axial ligand was provided indirectly by the observation of anomalously low values for the4oxidation-state marker mode, especially in the ferrous form, where it occurs near 1,346 cm1, approximately 10 cm1below its value for histidine-ligated ferroheme proteins. More importantly, direct confirmation of this linkage was provided by an impressive RR study, employing both 54Fe-labeled protoheme and 34S-labeled protein,24 documenting the existence of a (Fe–S) stretching mode in the ferric high-spin, substrate-bound form, which is sensitive to both isotopic labels. In fact, in an elegant study in 2002, Champion and co-workers have used RR spectroscopy to monitor changes in the (Fe–S) mode which reflect perturbations in this key Fe–S linkage brought about by interactions with native and modified redox partners.52
Early RR studies of the O2adduct, also reported by Champion and co-workers,51established its proper formulation as a ferriheme-superoxo species by observation of a strongly enhanced (16O–16O) stretching mode occurring at 1,140 cm1 (1,074 cm1 for the corresponding 18O2
adduct), a value typical of superoxo complexes. Later studies, reported by the Champion research group53and others,54identify the(Fe–O) stretching mode as a relatively weak16O/18O-sensitive band occurring near 540 cm1, a value also consistent with the suggested ferric superoxo formulation.
Given the impressively demonstrated utility of the RR technique as an exquisite probe of the active site structures of these cytochrome P450 species, it is anticipated that it will play a major role in defining the structure of subsequent intermediates in the enzymatic cycle of this class of proteins. However, for such expectations to be realized, it will be necessary to devise effective strategies to trap these highly reactive species, or to prolong their lifetime to an extent sufficient to permit acquisition of their RR spectra.
1250 1350 1450 1550 1650
1337 1378 1396 1428 1470 1508 1558 1582 1600 1620 1631 1639
1299 1318 1358 1379 1402 1429 1456 1502 1546 1571
1604 1615
1636
HRP CMP I
HRP CMP II
λexc. = 3638 Å
1544
Raman shift (cm–1)
Figure 7 High-frequency resonance Raman spectra of horseradish peroxidase compound I-HRP CMP I and compound II-HRP CMP II (adapted from ref. 47).