Defining resonance Raman spectral responses to substrate binding by cytochrome P450 from Pseudomonas putida.
Resonance Raman spectra are reported for substrate-free and camphor-bound cytochrome P450cam and its isotopically labeled analogues that have been reconstituted with protoheme derivatives that bear -CD(3) groups at the 1, 3, 5, and 8-positions (d12-protoheme) or deuterated methine carbons (d4-protoheme). In agreement with previous studies of this and similar enzymes, substrate binding induces changes in the high frequency and low frequency spectral regions, with the most dramatic effect in the low frequency region being activation of a new mode near 367 cm(-1). This substrate-activated mode had been previously assigned as a second "propionate bending" mode (Chen et al., Biochemistry, 2004, 43, 1798-1808), arising in addition to the single propionate bending mode observed for the substrate-free form at 380 cm(-1). In this work, this newly activated mode is observed to shift by 8 cm(-1) to lower frequency in the d12-protoheme reconstituted enzyme (i.e., the same shift as that observed for the higher frequency "propionate bending" mode) and is therefore consistent with the suggested assignment. However, the newly acquired data for the d4-protoheme substituted analogue also support an earlier alternate suggestion (Deng et al., Biochemistry, 1999, 38, 13699-13706) that substrate binding activates several heme out-of-plane modes, one of which (gamma(6)) is accidentally degenerate with the 367 cm(-1) propionate bending mode. Finally, the study of the enzyme reconstituted with the protoheme-d4, which shifts the macrocycle nu(10) mode, has now allowed a definitive identification of the vinyl C=C stretching modes.
Mak
Piotr J
PJ
Department of Chemistry, Marquette University, Milwaukee, WI 53233, USA.
Kaluka
Daniel
D
Manyumwa
Munyaradzi Edith
ME
Zhang
Haiqing
H
Deng
Tianjing
T
Kincaid
James R
JR
D001426:Bacterial Proteins / Q000737:chemistry*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D016958:Pseudomonas putida / Q000201:enzymology*; D013059:Spectrum Analysis, Raman / Q000379:methods*
D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System