Accommodating a Nonconservative Internal Mutation by Water-Mediated Hydrogen Bonding between β-Sheet Strands: A Comparison of Human and Rat Type B (Mitochondrial) Cytochrome b5

摘要

Mammalian type B (mitochondrial) b5 cytochromes exhibitngreater amino acid sequence diversity than their type A (microsomal) counter-nparts, as exemplified by the type B proteins from human (hCYB5B) and ratn(rCYB5B). The comparison of X-ray crystal structures of hCYB5B and rCYB5Bnreported herein reveals a striking difference in packing involving the five-strandnβ-sheet, which can be attributed to fully buried residue 21 in strand β4. Thengreater bulk of Leu21 in hCYB5B in comparison to that of Thr21 in rCYB5Bnresults in a substantial displacement of the first two residues in β5, andnconsequent loss of two of the three hydrogen bonds between β5 and β4.nHydrogen bonding between the residues is instead mediated by two well-nordered, fully buried water molecules. In a 10 ns molecular dynamics simulation,none of the buried water molecules in the hCYB5B structure exchanged readilynwith solvent via intermediates having three water molecules sandwichednbetween β4 and β5. When the buried water molecules were removed prior to a second 10 ns simulation, β4 and β5 formednpersistent hydrogen bonds identical to those in rCYB5B, but the Leu21 side chain was forced to adopt a rarely observednconformation. Despite the apparently greater ease of access of water to the interior of hCYB5B than of rCYB5B suggested by thesenobservations, the two proteins exhibit virtually identical stability, dynamic, and redox properties. The results provide new insight intonthe factors stabilizing the cytochrome b5 fold.