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 (TRS2) 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 H2O/D2O 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 v(Fe-CO)/v(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 (v(CO) = 2138 cm(-1)). The TRS2-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.