Unusual Spectroscopic and Ligand Binding Properties of the Cytochrome P450-Flavodoxin Fusion Enzyme XplA

Unusual Cytochrome P450 Enzymes and Reactions

“…XplA has unusual properties, including unusually weak binding of the FMN cofactor and the absence of a phylogenetically conserved Ser/Thr residue in the P450 I helix. The latter structural deviation is consistent with a primarily reductive role for XplA because the Ser/Thr residue is implicated in hydrogen bonding and/or proton transfer to iron-bound oxygen in the normal P450 catalytic cycle (82).…”

Structure and Biochemical Properties of the Alkene Producing Cytochrome P450 OleTJE (CYP152L1) from the Jeotgalicoccus sp. 8456 Bacterium

“…In the visible (heme Q-band) region, a single, slightly asymmetric feature is seen at ϳ540 nm. The blue shift of the Soret spectrum on reduction indicates substantial retention of cysteine thiolate proximal coordination in the OleT JE ferrous state, and the spectral maxima are similar to those features seen in, for example, the well characterized Pseudomonas putida camphor hydroxylase P450cam (CYP101A1, 411 and 540 nm) and in the explosive degrading P450 XplA from Rhodococcus rhodochrous strain 11Y (CYP177A1, 408 and 542 nm) (23,29). The addition of carbon monoxide to anaerobically reduced OleT JE produced a characteristic P450 heme spectrum with the Soret band red-shifted to 449 nm and a Q-band feature at 551 nm.…”

“…The 423 nm peak arises from distal ligation of DTT thiol to OleT JE heme iron, whereas the outer peaks result from a split (hyperporphyrin) Soret spectrum in which DTT thiolate ligates the iron (36,37). Comparable spectral maxima are at 374, 423.5, and 453.5 nm for XplA (23). In XplA, the intensities of the three absorbance bands are quite similar, but in OleT JE , the outer bands are much weaker than the 423 nm feature, suggesting that DTT favors heme ligation in the thiol state under the conditions used.…”

“…For substrate-bound OleT JE , the titration was done under the same conditions, following the addition of arachidic acid (from a 32 mM stock in 80% EtOH, 20% Triton X-100) until no further conversion of the heme iron to the HS heme state was observed (ϳ12 M arachidic acid). Mediators were added to expedite electronic equilibration in the system (2 M phenazine methosulfate, 7 M 2-hydroxy-1,4-naphthoquinone, 0.3 M methyl viologen, and 1 M benzyl viologen to mediate in the range from ϩ100 to Ϫ480 mV versus normal hydrogen electrode (NHE)) and data fitting (using the Nernst equation), and analysis was done as described previously (21)(22)(23).…”

“…The binding of DTT to OleT JE was also analyzed in view of the report from Rude et al (18), which indicated that DTT could support OleT JE fatty acid decarboxylase activity by producing H 2 O 2 under aerobic conditions in the presence of the P450 heme iron (18,35). However, in previous studies, we showed that DTT coordinated the heme iron in the explosive degrading XplA P450 (23). DTT is known to bind P450 heme iron, and ligation is feasible in both DTT thiol and thiolate forms (36,37).…”