Defining resonance Raman spectral responses to substrate binding by cytochrome P450 from Pseudomonas putida

Abstract

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 -CD3 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, Z.; Ost, T. W. B.; Schelvis, J. P. M. 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 the present 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, T. J.; Proniewicz, L. M.; Kincaid, J. R.; Yeom, H.; Macdonald, I. D. G.; Sligar, S. G. Biochemistry 1999 38, 13699−13706) that substrate binding activates several heme out-of-plane modes, one of which ( γ6) 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 ν10 mode, has now allowed a definitive identification of the vinyl C=C stretching modes.

Defining CYP3A4 Structural Responses to Substrate Binding. Raman Spectroscopic Studies of a Nanodisc-Incorporated Mammalian Cytochrome P450

“…It is noted that the HS components of the ν 2 and ν 10 modes are not readily observed, because of overlaps with other modes (i.e., ν 37 and ν(C=C) vinyl modes). Most interestingly, it can be seen that the addition of substrate (testosterone in a 10-fold excess) to this conventionally solubilized protein does not substantially affect the spin state distribution of the enzyme (trace B), a result in stark contrast to results normally obtained for most soluble bacterial cytochromes P450, where binding of typical substrates causes conversion of an almost pure LS state to population having 90-98 % HS component 25,32. The spectroscopic behavior observed here for these detergent-solubilized proteins suggests that the “substrate-free” sample shows some conversion to HS state owing to the presence of the Emulgen 913, which has been suggested to be a substrate for mammalian P450s,64 but that the added TST substrate is to some extent blocked from entering the active site, as reflected in the total absence of further conversion to the HS state.…”

“…The spectra of the substrate-free enzyme, as well as that bound with 10 molar equivalents of testosterone, are shown in Figure 3 (traces A and B, “CM” stands for “conventional media”, as opposed to nanodisc-incorporated proteins that are the main subject of this paper). The so-called spin state marker bands in these spectra are expected to occur near 1500 (v 3 ), 1590 (ν 2 ) and 1640 cm -1 (ν 10 ) for the LS state and near 1490, 1570 and 1630 cm -1 for the HS state 18,19,23-25,32. As can be readily seen in the top trace, the ν 3 spin state marker bands of the substrate-free state indicate the presence of substantial amounts of both low (1502 cm -1 ) and high (1488 cm -1 ) spin components; however, it is noted that the intensity ratio cannot be assumed to be directly proportional to the spin state populations owing to differences in inherent RR cross-sections.…”

“…In order to document the fractional population of these multiple Fe-CO conformers, the low frequency RR spectra of ferrous CO adducts were analyzed with Grams 32/AI software using a peak fitting procedure, employing 50/50% Gaussian/Lorentzian functions 64. The fitting was performed in a manner that restricts the number of peaks to the minimum, noting that this region also contains a well known heme mode 24,25. Thus, in order to obtain satisfactory fitting of Fe-CO envelope, it was necessary to include this feature.…”

“…High frequency marker modes respond to changes in oxidation- or spin-state of the central iron in well-established and documented ways, while low frequency modes indicate changes in protein interactions with the heme periphery 20-25. This is important, because the presence of the propionic acid and (potentially conjugated) vinyl peripheral substituents have long been considered as possibly important structural determinants of heme reactivity, whose influence may be sensitively manipulated by protein-heme interactions 23,26-28.…”

The Crystal Structure of Cytochrome P450 4B1 (CYP4B1) Monooxygenase Complexed with Octane Discloses Several Structural Adaptations for ω-Hydroxylation

“…Mammalian peroxidases exhibit similar covalent linkages between acidic side chains of the peroxidases and the 5-methyl and 8-methyl carbons of their heme prosthetic groups, and similar out-of-plane distortions of their heme prosthetic groups are evident in structures of these enzymes (42). Resonance Raman studies suggest that the out-of-plane distortions of the heme in lactoperoxidase are relaxed when the enzyme is cleaved by limited proteolysis suggesting that protein interactions with the heme in the enzyme's active site induce the distortion (43).…”