{'fraction_non_alphanumeric': 0.04495912806539509, 'fraction_numerical': 0.027247956403269755, 'mean_word_length': 5.707762557077626, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Aluminum‐substituted heme domain of P450BM‐3 (BMP): Introducing a heme‐derived fluorescent probe for studies of substrate binding and protein–protein interactions in cytochromes P450

We describe a novel technique for heme removal and replacement in the heme domain of P450BM‐3 (BMP). The method was applied to obtain the aluminum‐protoporphyrin IX (Al‐PP) substituted derivative of BMP (Al‐BMP). The overall yield of the purified Al‐BMP was about 15% as related to the initial amount of the hemeprotein. Al‐BMP possesses extensive fluorescence in the 550–650 nm region with excitation in the porphyrin absorbance bands. The protein was shown to bind substrates of P450BM‐3 (palmitic, arachidonic, and cis‐parinaric acids) with affinities similar to those of the native enzyme (3–6 µM). However, the substrate‐induced changes in fluorescence of Al‐PP reveal the existence of a second, low‐affinity substrate‐binding site, which cannot be detected by the spin shift in the native, heme‐containing BMP. Using fluorescence resonance energy transfer, we have demonstrated that Al‐BMP forms a complex with the flavoprotein domain of P450BM‐3 labeled with 7‐ethylamino‐3‐(4'‐maleimidylphenyl)‐4‐methylcoumarin maleimide, revealing the affinity similar to that of native BMP (Kd = 5 µM at 0.06 M ionic strength). Therefore, aluminum‐substituted BMP may serve as a valuable tool in studies on the mechanisms of interactions of P450s with their substrates and protein partners.