title: Aldoxime dehydratase: probing the heme environment involved in the synthesis of the carbon-nitrogen triple bond.
abstract: Fourier transform infrared (FTIR) spectra, "light" minus "dark" difference FTIR spectra, and time-resolved step-scan (TRS(2)) FTIR spectra are reported for carbonmonoxy aldoxime dehydratase. Two C-O modes of heme at 1945 and 1964 cm(-1) have been identified and remained unchanged in H(2)O/D(2)O exchange and in the pH 5.6-8.5 range, suggesting the presence of two conformations at the active site. The observed C-O frequencies are 5 and 16 cm(-1) lower and higher, respectively, than that obtained previously (Oinuma, K.-I.; et al. FEBS Lett.2004, 568, 44-48). We suggest that the strength of the Fe-His bond and the neutralization of the negatively charged propionate groups modulate the ν(Fe-CO)/ν(CO) back-bonding correlation. The "light" minus "dark" difference FTIR spectra indicate that the heme propionates are in both the protonated and deprotonated forms, and the photolyzed CO becomes trapped within a ligand docking site (ν(CO) = 2138 cm(-1)). The TRS(2)-FTIR spectra show that the rate of recombination of CO to the heme is k(1945 cm(-1)) = 126 ± 20 s(-1) and k(1964 cm(-1)) = 122 ± 20 s(-1) at pH 5.6, and k(1945 cm(-1)) = 148 ± 30 s(-1) and k(1964 cm(-1)) = 158 ± 32 s(-1) at pH 8.5. The rate of decay of the heme propionate vibrations is on a time scale coincident with the rate of rebinding, suggesting that there is a coupling between ligation dynamics in the distal heme environment and the environment sensed by the heme propionates. The implications of these results with respect to the proximal His-Fe heme environment including the propionates and the positively charged or proton-donating residues in the distal pocket which are crucial for the synthesis of nitriles are discussed.
authors.affiliation: Department of Chemistry, University of Cyprus, Nicosia, Cyprus. effiep@ucy.ac.cy
authors.forename: Eftychia
authors.initials: E
authors.lastname: Pinakoulaki
authors.forename: Constantinos
authors.initials: C
authors.lastname: Koutsoupakis
authors.forename: Hitomi
authors.initials: H
authors.lastname: Sawai
authors.forename: Andrea
authors.initials: A
authors.lastname: Pavlou
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Kato
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Asano
authors.forename: Shigetoshi
authors.initials: S
authors.lastname: Aono
mesh_terms: D002244:Carbon / Q000737:chemistry* / Q000378:metabolism; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D009584:Nitrogen / Q000737:chemistry* / Q000378:metabolism; D011422:Propionates / Q000737:chemistry / Q000378:metabolism; D011522:Protons; D012240:Rhodococcus / Q000737:chemistry / Q000201:enzymology*; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D008024:Ligands; D011422:Propionates; D011522:Protons; D006418:Heme; D002244:Carbon; D002248:Carbon Monoxide; D006836:Hydro-Lyases; C412124:aldoxime dehydratase; D009584:Nitrogen