UV resonance Raman probe of heme‐bound imidazole established by ¹⁵N‐labeling of hemoglobin

Abstract

Recent studies of Cu, Zn superoxide dismutase, and of zinc-finger peptides have established that histidine ligands can be detected in ultraviolet resonance Raman (UVRR) spectra, following NH/D exchange of the imidazole. UVRR spectroscopy therefore offers promise for monitoring histidine ligation in heme proteins. In this work, we characterize heme-bound histidine UVRR bands for N-acetyl-microperoxidase-8 (MP-8) and microperoxidase-11 (MP-11), and also for hemoglobin (Hb). The Hb UVRR spectra are dominated by tyrosine and tryptophan contributions, but a band appears at 1340 cm -1 in D 2 O solution, which is assigned to a mode of Fe-bound imidazole. This band shifted 24 cm -1 in protein which was labeled with 15 N via expression of the Hb gene in E. coli grown on 15 NH 4 + . In MP-11, the position of this band is insensitive to ligation or oxidation state changes, but it is 2 cm -1 lower in deoxyHb than in the CO adduct. This shift may reflect mechanical forces on the proximal histidine in the T state, and/or changes in its H-bonding.

Perturbations of Aromatic Amino Acids Are Associated with Iron Cluster Assembly in Ribonucleotide Reductase

“…As shown in Figure S4D for histidine in vitro , 15 N labeling of histidine results in a 26 cm −1 downshift of the 1408 cm −1 band. This downshift is expected from previous studies 59 Figures 6A and B. show a similar downshift (1405 (+) to 1381 (+) cm −1 ) in the 15 N 3 histidine labeled Apo-β2-minus-Met-β2 spectrum.…”

Assignment of the 1511 cm−1 UV resonance Raman marker band of hemoglobin to tryptophan

“…4). In this experiment 15‐N was incorporated into Hb by expressing the protein in bacteria grown on 15‐NH, and spectra were recorded in D 2 O in order to intensify the histidine signals12; a large 15‐N shift was seen for the 1407 cm −1 band that arises from protonated histidine residues 14. The 2 cm −1 downshift in 2×W18 is consistent with the 1 cm −1 shift, which was reported for the fundamental 15…”

“…Solutions (0.5 m M in heme) were prepared in 50 m M phosphate buffer at pH 7.4; NaClO 4 (0.2 M ) was added to the sample as an internal standard. The preparation of 15‐N labeled recombinant Hb is described elsewhere 12. For the H/D exchange experiments the Hb was exchanged into 50 m M phosphate buffer in D 2 O (pD 7.4) with 0.2 M NaClO 4 as the Raman standard.…”

Raman structural markers of tryptophan and histidine side chains in proteins

“…15,69 Because metal coordination is one of the most important properties of His and many proteins contain metal-ligated His residues, the Raman spectra of metalloproteins provide the basis for testing and modifying the proposed metal coordination markers. Such proteins include Cu,Zn-superoxide dismutase (SOD), 15,69 -71 hemoproteins, 60,72 and plastocyanin. 61 The Raman spectra of Zn-finger peptides, 73,74 a prion protein, 75,76 amyloid ␤-peptides, 77,78 an influenza virus matrix protein, 79 and a small bioactive peptide 80 have also been studied with special attention to the metal binding mode of His.…”

“…69,71 The utility of the 1440 -1434 cm for N -metal-bound His residues in proteins: two His residues of a Zn finger 74 and the Febound proximal His residues of hemoglobin and microperoxidases. 72 However, UV Raman bands have also been found in the 1365-1344 cm Ϫ1 region for plastocyanin, 61 of which two His residues are bound to a Cu ion via N but not via N , and for Cu-depleted SOD, 71 in which the remaining Zn ion is expected to bind to the N atoms of three His residues in the active site. The imidazolate bridge ([N -M, N -MЈ]) is characterized by a strong Raman band that is attributable to a stretch mode of the C 2 ON OC 4 linkage 69,81 at 1292-1282 cm Ϫ1 in both visible and UV Raman spectra.…”