Identification by proton nuclear magnetic resonance of the histidines in cytochrome b5 modified by diethyl pyrocarbonate

Abstract

Diethyl pyrocarbonate (DEP) is an electrophilic reagent that is used to modify reversibly the histidine residues of proteins. Unfortunately, the lability of the acylated histidine adduct usually does not permit the isolation and identification of the modified histidine. By use of 500-MHz proton NMR spectroscopy, it has been possible to identify the C-H resonances of the nonaxial histidines of trypsin-solubilized bovine, rabbit, and porcine cytochrome b5 and therefore observe the interaction of DEP with specific histidine residues of cytochrome b5. In addition, the pKa of the peripheral histidines of bovine and rabbit cytochrome b5 have been measured in D2O. In the bovine protein it was found that the histidines are modified sequentially with increasing DEP concentration in the order His-26 greater than His-15 greater than His-80. This order is maintained in the rabbit protein with the following additions: His-26 approximately His-27 greater than His-15 greater than or equal to His-17 greater than His-80. The relative reactivity of the peripheral histidines with DEP was rationalized by considering three of their characteristics: (1) the pKa of the histidine, (2) the fraction of the side chain exposed to the solvent, and (3) the hydrogen-bond interactions of the imidazole ring.

Proton linkage of complex formation between cytochrome c and cytochrome b5: electrostatic consequences of protein-protein interactions

“…In particular, we note that the pKa of cytochrome b, residue His-26 that we have implicated above as undergoing a significant binding-induced pKa shift is also predicted to be acutely sensitive to ionic strength (Mauk et al, 1986;Altman et al, 1989).…”

Enhancing the thermal stability of mitochondrial cytochrome b5 by introducing a structural motif characteristic of the less stable microsomal isoform

“…Figure 9 shows two distinct patterns that are possible for the fully H-bonded Glu11-His15-Ser20 triad. NMR studies have shown that His15 has a pK a of $8.5 in bMc holo-b 5 (Altman et al, 1989), as well as in rat Mc holo-and apo-b 5 (Moore et al, 1991). It is therefore safe to assume that His15 in bMc apo-b 5 also has a pK a of $8.5, and is protonated at the pH of 7.0 utilized in our studies (as in Fig.…”

Histidine residues in rabbit liver microsomal cytochrome P-450 2B4 control electron transfer from NADPH-cytochrome P-450 reductase and cytochrome b5

“…Irrespective of whether P-450 2B4 was labelled to the extent of a statistical average of 3.5 or 11.2 carbethoxy groups introduced per polypeptide within the time frame of the experiment, biphasic kinetic patterns were observed ( Figures 1B and 1C). Unless the particular pK a values for the individual histidines or specificities in solvent accessibility accounted for this dualism, the slowreacting species might have participated in hydrogen bonding with proton donors [34] ; as is well known, the electrophilic DEPC molecule interferes preferentially with the unprotonated form of histidine [24]. From the tracings depicted in Figure 1(C), the proportions of fast-and slow-reacting material were computed as 35 and 65 % respectively, characterized by pseudo-firstorder rate constants of 0.3 s −" and 0.06 s −" .…”