Schulze H, Hoa G H, Helms V, Wade R C, Jung C
Max-Delbrück-Centrum für Molekulare Medizin Berlin-Buch, Berlin, Germany.
Biochemistry. 1996 Nov 12;35(45):14127-38. doi: 10.1021/bi9527303.
A comparative study of the enantiomeric substrate [(1R)-camphor- and (1S)-camphor)-bound cytochrome P-450cam concerns the spin-state equilibrium, substrate dissociation, the thermal unfolding of the protein structure, and the subconformer equilibria observed in the infrared spectra of the carbon monoxide (CO) complex of cytochrome P-450cam. The behavior of the different conformational equilibria in dependence on temperature, pressure, pH-value, cosolvent, and cation binding led us to suggest that (1S)-camphor is more loosely and less optimally bound in the heme pocket, which facilitates the access of solvent molecules into the heme-iron environment. The spin reaction volume difference measured using the high pressure technique is smaller by 16 +/- 9 cm3/mol for (1S)-camphor-bound P-450cam compared to the (1R)-camphor-bound P-450cam, which might indicate a higher water content in the protein and in the heme environment in the (1S)-camphor complex. The half-transition temperature of the thermal unfolding of 53.8 degrees C for the (1S)-camphor-bound oxidized cytochrome P-450cam is one degree lower than the value for the (1R)-camphor-bound protein (54.8 degrees C). In the reduced, CO-bound form of cytochrome P-450cam at 290 K the (1S)-camphor complex reveals another CO stretch vibration population distribution with slightly higher frequencies [1940.2 cm-1 (major band) and 1946.3 cm-1 (minor band)] compared to the (1R)-camphor complex [1939.7 cm-1 (major band) and 1930 cm-1 (minor band)]. A loosening of the contact between the iron-bound CO ligand and amino acids of the I-helix, probably induced by compensating effects of the increased water content, is suggested. Assuming the carbon monoxide complex as a model for the dioxygen complex, the more loosened binding of (1S)-camphor, therefore the increased water accessibility, and the weaker contact of the iron ligand to the I-helix might explain the higher amount of uncoupling of the cytochrome P-450 reaction cycle compared to that when (1R)-camphor is used as substrate.
对映体底物[（1R）-樟脑和（1S）-樟脑]结合的细胞色素P-450cam的比较研究涉及自旋态平衡、底物解离、蛋白质结构的热解折叠以及在细胞色素P-450cam的一氧化碳（CO）配合物的红外光谱中观察到的亚构象平衡。不同构象平衡随温度、压力、pH值、共溶剂和阳离子结合的变化行为使我们认为，（1S）-樟脑在血红素口袋中的结合较松散且不太理想，这有利于溶剂分子进入血红素-铁环境。与（1R）-樟脑结合的P-450cam相比，使用高压技术测得的（1S）-樟脑结合的P-450cam的自旋反应体积差小16±9 cm³/mol，这可能表明（1S）-樟脑配合物中蛋白质和血红素环境中的水含量较高。（1S）-樟脑结合的氧化型细胞色素P-450cam的热解折叠的半转变温度为53.8℃，比（1R）-樟脑结合的蛋白质的值（54.8℃）低1℃。在290K下细胞色素P-450cam的还原态、CO结合形式中，与（1R）-樟脑配合物[1939.7 cm⁻¹（主带）和1930 cm⁻¹（次带）]相比，（1S）-樟脑配合物显示出另一种频率略高的CO伸缩振动种群分布[1940.2 cm⁻¹（主带）和1946.3 cm⁻¹（次带）]。有人提出，铁结合的CO配体与I-螺旋氨基酸之间的接触可能因水含量增加的补偿作用而松弛。假设一氧化碳配合物为双氧配合物的模型，（1S）-樟脑结合较松散，因此水的可及性增加，以及铁配体与I-螺旋的接触较弱，这可能解释了与使用（1R）-樟脑作为底物时相比，细胞色素P-450反应循环的解偶联量更高的原因。
