Functions of fluctuation in the heme-binding loops of cytochrome b(5) revealed in the process of heme incorporation

Cytochrome b(5) (cyt b(5)) holds heme using two axial histidines, His63 and His39, that are located in the centers of the two heme-binding loops. The previous NMR study on the apo form of cyt b(5) (apocyt b(5)) revealed that the loop including His63 exhibits a larger fluctuation compared to the othe r loop including His39 [Falzone, C. J., Mayer, M. R., Whiteman, E. L., Moor e, C. D., and Lecomte, J. T. (1996) Biochemistry 35, 6519-6526]. To underst and the significance of the fluctuation, the heme association and dissociat ion rates of the two loops were compared using two mutants of cyt b(5) in w hich one of the axial histidines was replaced with leucine. It was demonstr ated that the fluctuating loop possesses a significantly slower heme dissoc iation rate and a faster heme association rate than the other loop. To furt her verify the importance of the fluctuating loop, the heme association pro cess of wild-type apocyt b(5) was investigated using optical absorption and CD spectroscopies. It was indicated that the process proceeds through the two pathways, and that the dominant pathway involves the initial coordinati on of His63 located in the fluctuating loop. The urea concentration depende ncy of the rate constants revealed that the folding of the fluctuating loop is associated with the coordination of His63. It was suggested that the fl uctuation enables the loop to have a larger heme-loop contact in the heme-b ound conformation. The fluctuating heme-binding loops might be useful for t he artificial design of heme-binding proteins.