Molecular dynamics simulation studies of cytochrome b₅ from outer mitochondrial and microsomal membrane

Abstract

Two forms of cytochrome b(5) have been identified, associated with the outer membrane of liver mitochondria (OM cyt b(5)) and with the membrane of the endoplasmic reticulum (microsomal, Mc cyt b(5)). These proteins have very similar structures, but differ significantly in physical properties, with the OM cyt b(5) exhibiting a more negative reduction potential, higher stability, and stronger interactions with the heme. We perform molecular dynamics simulations to probe the structures and fluctuations of the two proteins in solution, to help explain the observed physical differences. We find that the structures of the two proteins, highly similar in the crystal, differ in position of a surface loop involving residues 49-51 in solution. Hydrophobic residues Ala-18, Ile-32, Leu-36, and Leu-47 tend to cluster together on the surface of rat OM cyt b(5), blocking water access to the protein interior. In bovine Mc cyt b(5), two of these positions, Ser-18 and Arg-47, are occupied by hydrophilic residues. This leads to breaking the hydrophobic cluster and allowing the protein to occupy a more open conformation. A measure of this structural transition is the opening of a cleft on the protein surface, which is 5 A wider in the OM cyt b(5) simulation compared to the Mc form. The OM protein also appears to have a more compact hydrophobic core in its beta-sheet region. These effects may be used to explain observed stability differences between the two proteins.

Influence of point mutations on the flexibility of cytochrome b5: Molecular dynamics simulations of holoproteins

“…In our group, independent MD simulations of 1-ns length, differing from those of Storch and Daggett in starting structures, force field, and the presence of counterions, were carried out for both rat OM and bovine Mc b 5 . 9,14 In many respects, OM and Mc b 5 behaved similarly in our trajectories. The exception was the position of a surface loop comprising residues 49-51.…”

“…20 The starting coordinates and simulation setup for the rat OM and bovine Mc b 5 have been described previously. 9,14 Starting conformations of the two mutants of OM b 5 were structures obtained from the Xray coordinates deposited in the Protein Data Bank (PDB): chain D from file 1ICC for the triple mutant A18S/I32L/ L47R (OM 3M ) 9 and chain B from file 1LJ0 for the quintuple mutant A18S/I25L/I32L/L47R/L71S (OM 5M ). 10 The starting coordinates for rat Mc b 5 were obtained from the NMRdetermined structure 1AW3.…”

“…13 Our extended MD simulations found analogous cleft opening motions in bovine Mc b 5 that were not evident in the previous shorter simulation (Figure 4). 9,14 The remaining proteins exhibited fluctuations in cleft size but no large-scale transitions. As suggested by Storch and Daggett, such differences in structural flexibility may have functional significance, modulating the ability of the cytochromes to interact with different partners.…”

“…13 However, our relatively short simulations suggested a permanent population of the closed structure by OM and the open structure by Mc b 5 , rather than open-closed fluctuations. 14 In this work we present results of MD simulations aimed at explaining the microscopic effects responsible for the differences in physical properties between OM and Mc b 5 . To study the contribution of the two hydrophobic patches to the properties of rat OM b 5 , we have performed simulations of $9-ns length for the heme-binding domains of OM 3M and OM…”

Molecular modeling of cytochrome b 5 with a single cytochrome c-like thioether linkage

“…To date, plenty of theoretical studies have been performed for cyt b 5 [24][25][26][27][28][29][30][31][32], cyt b 562 [33][34][35][36], and cyt c [37][38][39]; these studies have aimed to gain structural insights, as well as to elucidate the mechanisms of holoprotein maturation. Using molecular modeling in combination with MD simulations, we recently studied the structural consequences of introducing a histidine tag, the (His) 6 tag, to the N-terminus of bovine Mc cyt b 5 [32].…”