Aldoxime dehydratase: probing the heme environment involved in the synthesis of the carbon-nitrogen triple bond.

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.