Steady‐State and Picosecond‐Time‐Resolved Fluorenscence Studies on the Recombinant Heme Domain of Bacillus megaterium Cytochrome P‐450

Abstract

The conformational changes associated with the interaction of sodium laurate with the recombinant heme domain for cytochrome P-450BM3 have been investigated by steady-state and picosecond-timeresolved fluorescence spectroscopy. The steady-state quenching experiments show that while all the five tryptophan residues are accessible to acrylamide in the free enzyme as well as the enzyme . substrate complex, the number of tryptophan residues accessible to ionic quenchers decreases on interaction of the substrate with the enzyme. This indicates that some of the tryptophan residues move towards the core of the protein on interaction with the substrate. The number of tryptophan residues accessible to the solvent as determined by the calculation of the solvent-accessible area for the free enzyme agrees with the values obtained by the quenching experiments.The time-resolved fluorescence studies carried out by means of the time-correlated single-photoncounting technique show that the fluorescence-decay curve is best fitted to a three-exponential model (0.2, 1.0 and 5.4 ns). Lifetime distributions, as recovered by the maximum-entropy method, agree with the discrete exponential model. The binding of the substrate does not lead to any significant change in the lifetime components of the enzyme, indicating that the tryptophan residues are possibly away from the substrate-binding domain. The decay-associated emission spectra and the magnitudes of amplitude of different lifetimes indicate that the shortest lifetime component ( 7 , ) originates from the three tryptophan residues that are completely or partially accessible to the solvent, and t, originates from the tryptophan residues that are buried in the core of the enzyme and not accessible to the solvent. X-ray crystallographic data and solvent-acessible-area calculations have been used to identify these residues.

Structural-Functional Characterization and Physiological Significance of Ferredoxin-NADP+ Reductase from Xanthomonas axonopodis pv. citri

“…Measurement of FNR fluorescence quenching by titration with a dynamic quencher can be used to analyze the accessibility of FAD [35], [36]. Using this experimental approach, we detected that the FAD isoalloxazine was more exposed to the solvent in Xac -FNR and in pea-FNR than in Ec -FNR (Figure 4C).…”

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

“…Such a transition is likely to be promoted by a temperature-steered structural rearrangement: Substrate may slide from a position directly atop the heme, corresponding to high-spin protein, to a more distant one related to the low-spin conformer [71,72]. In fact, tryptophan residue(s) along with F87 have been shown to be instrumental to facile conformational switching elicited by substrate liganding to CYP102A1 [73,74]. The sum of these findings is suggestive of a plastic, compressible active-site architecture affording considerable substrate mobility [75].…”

“…Thus, employing second-derivative along with temperature jump spectroscopy, substrate binding to CYP101A1 and CYP2B5 was detected to perturb the microenvironment of activesite tyrosine(s) such as to change dynamic fluctuations of the aqueduct governing accessibility of bulk solvent [37,39,41,184]. Steadystate fluorescence as well as one-and two-dimensional NMR measurements with isotopically-enriched CYP102A1 suggested a general substrate-induced structural rearrangement in the enzyme pocket [71,73,74]; these findings were in accord with results from replica exchange molecular dynamics studies [72]. Similarly, application of surface-enhanced Raman scattering served to disclose distortion of the heme vicinity of CYP2B4 upon liganding of sterically distinct substrates [185], while CD-spectral analyses in the far-ultraviolet region pointed at more global modifications of the secondary structural features of CYP2B4 and CYP11A1 [186,187].…”

Atomic force microscopy monitoring of the temperature dependence of the cytochrome BM3 oligomeric state

“…BM3 has 12 Trp residues and 35 Tyr residues (according to the known structure), including 5 Trp residues in the heme domain [20], 7 Trp residues in the reductase domain, and flavin binding Trp 574 residue [21]. Therefore, the influence of the temperature on the BM3 structure was studied using the fluorescence spectra of both the aro matic amino acid residues and flavin groups.…”