Direct Infrared Detection of the Covalently Ring Linked His−Tyr Structure in the Active Site of the Heme−Copper Oxidases

Abstract

Infrared spectroscopy, isotopic labeling ([(15)N(delta,epsilon)]histidine and ring-deuterated tyrosine), synthetic model studies, and normal mode calculations are employed to search for the spectroscopic signatures of the unique, covalently linked (His N(epsilon)-C(epsilon) Tyr) biring structure in the heme-copper oxidases. The specific enzyme examined is the cytochrome bo(3) quinol oxidase of E. coli. Infrared features of histidine and tyrosine are identified in the frequency regions of imidazole and phenol ring stretching modes (1350-1650 cm(-1)) and C-H and N-H stretching modes as well as overtones and combinations (>3000 cm(-1)). Two of these, at ca. 1480 and 1550 cm(-1), and their combination tones between 3010 and 3040 cm(-1), are definitively identified with the biring structure involving H284 and Y288 in the E. coli enzyme. Studies of a synthetic analogue of the H-Y structure, 4-methylimidazole covalently linked to p-cresol, show that a feature near 1540 cm(-1) is unique to the biring structure and is absent from the infrared spectrum of 4-methylimidazole or p-cresol alone. This feature is readily detectable by infrared difference techniques, and offers a direct spectroscopic probe for potential radical production involving the H-Y structure in the O(2) reduction cycle of the oxidases.

Probing Protonation/Deprotonation of Tyrosine Residues in Cytochrome ba3 Oxidase from Thermus thermophilus by Time-resolved Step-scan Fourier Transform Infrared Spectroscopy

“…pK a of the Tyrosine Residue-Intensity changes and frequency shifts of side chains and backbone structures have been observed in the FTIR difference spectra of heme-copper oxidases as the result of an electrochemical perturbation (oxidized minus reduced) of the metal centers at room temperature or of the perturbation induced by the photodissociation of CO bound to the heme at 80 K, at which the CO binds irreversibly to Cu B (18,19,23,24). In the latter case, the perturbation induced by the photodissociated CO exerts its main effect on the environment of the binuclear center.…”

“…FTIR spectroscopy has proven to be a very powerful technique in studying changes at the level of individual amino acids during protein action (18,19). FTIR studies of a His-Tyr-OH model compound, 2-imidazol-1-yl-4-methylphenol, have demonstrated that the 7a (CO) and ␦(COH) modes in the deprotonated form of His-Tyr-O Ϫ shift from 1268 cm Ϫ1 (protonated) to 1301 cm Ϫ1 (18).…”

“…With the purpose of identifying His-Tyr-OH modes in cytochrome bo 3 oxidase, Woodruff and co-workers (19) applied low-temperature FTIR difference spectroscopy to the enzyme and FTIR spectroscopy to the His-Tyr-OH model compound. They detected the fundamental His N ⑀ -C ⑀ Tyr mode and its combination at 1549 and 3033 cm Ϫ1 , respectively (19). In addition to the dioxygen activation and reduction and the oxygenation of CO mechanisms, it is crucial to determine the intermediate protein structures subsequent to ligand binding/ release and discrimination (20).…”

Identification of a histidine-tyrosine cross-link in the active site of the cbb 3 -type cytochrome c oxidase from Rhodobacter sphaeroides

“…The only difference is the lack of the OH group in the aromatic ring while the atoms forming the link are still present. Because the metal centers of the active site can provide only three electrons out of the four needed in the full reduction of molecular oxygen to water, tyrosine has been suggested to have a key role in the O 2 reduction chemistry; the hydroxyl group of the tyrosine may donate both an electron and a proton to break the O-O bond, thereby forming a neutral tyrosine radical in the P M state of the catalytic cycle (22)(23)(24). This may avoid production of reactive oxygen species and might initialize the formation of the crosslink itself.…”

Redox-induced Protein Structural Changes in Cytochrome bo Revealed by Fourier Transform Infrared Spectroscopy and [13C]Tyr Labeling

“…(77). Recently, FTIR studies on synthetic models for the His-Tyr cross-link and oxidases demonstrated that a prominent band around 1540 cm Ϫ1 is assignable to the protonated form of the cross-linked tyrosine (42,51,55,78). The spectral comparison of photo-reduced minus air-oxidized forms for [4-13 C]Tyr-labeled and unlabeled cytochrome bo in the 1180 -1330 cm Ϫ1 region and at around 1540 cm Ϫ1 showed no isotope-sensitive spectral changes in the typical frequency region of tyrosine side chains and for the cross-linked tyrosine (not shown).…”