As part of a larger effort to engineer the stability and hemin-binding properties of microsomal (Mc) cytochromes b(5) into rat liver outer mitochondrial membrane (OM) cytochrome (cyt) b(5), several mutants of rat OM cyt b(5) were prepared to study the effect of gradual and complete elimination of two extended hydrophobic networks, which are present in the structure of the mitochondrial protein and are absent in the structure of mammalian Mc cytochromes b(5). One of the hydrophobic networks, identified in a previous study [Altuve, A., Silchenko, S., Lee, K.-H., Kuczera, K., Terzyan, S., Zhang, X., Benson, D. R., and Rivera, M. (2001) Biochemistry 40, 9469-9483], encompasses the side chains of Ala-18, Ile-32, Leu-36, and Leu-47, whereas a second hydrophobic network, identified as part of this work, encompasses the side chains of Ile-25, Phe-58, Leu-71, and the heme. The X-ray structure of the A18S/I25L/I32L/L47R/L71S quintuple mutant of rat OM cyt b(5) demonstrates that both hydrophobic networks have been eliminated and that the corresponding structural elements of the Mc isoform have been introduced. The stability of the rat OM mutant proteins studied was found to decrease in the order wild type > I25L > A18S/I32L/L47R > L71S > A18S/I32L/L47R/L71S > 18S/I25L/I32L/L47R/L71S, indicating that the two hydrophobic networks do indeed contribute to the high stability of rat OM cyt b(5) relative to the bovine Mc isoform. Surprisingly, the quintuple mutant of rat OM cyt b(5) is less stable than bovine Mc cyt b(5), even though the former exhibits significantly slower rates of hemin release and hemin reorientation at pH 7.0. However, at pH 5.0 the bovine Mc and rat OM quintuple mutant proteins release hemin at comparable rates, suggesting that one or both of the His axial ligands in the rat OM protein are more resistant to protonation under physiological conditions. Results obtained from chemical denaturation experiments conducted with the apoproteins demonstrated that mutants containing L71S are significantly less stable than bovine Mc apocyt b(5), strongly suggesting that Leu-71 plays a pivotal role in the stabilization of rat OM apocyt b(5), presumably via hydrophobic interactions with Ile-25 and Phe-58. Because comparable interactions are absent in bovine Mc apocyt b(5), which contains Ser at position 71, it must resort to different interactions to stabilize its fold, thus highlighting yet another difference between rat OM and bovine Mc cyt b(5). During the course of these investigations we also discovered that rat OM cyt b(5) can be made to strongly favor hemin orientational isomer A (I32L) or isomer B (L71S) with a single point mutation and that release of hemin orientational isomers A and B can be kinetically resolved in certain rat OM mutants.

The microsomal (Mc) and mitochondrial (OM) isoforms of mammalian cytochrome b 5 are the products of different genes, which likely arose via duplication of a primordial gene and subsequent functional divergence. Despite sharing essentially identical folds, heme-polypeptide interactions are stronger in OM b 5 s than in Mc b 5 s due to the presence of two conserved patches of hydrophobic amino acid side chains in the OM heme binding pockets. This is of fundamental interest in terms of understanding heme protein structurefunction relationships, because stronger heme-polypeptide interactions in OM b 5 s in comparison to Mc b 5 s may represent a key source of their more negative reduction potentials. Herein we provide evidence that interactions amongst the amino acid side chains contributing to the hydrophobic patches in rat OM (rOM) b 5 persist when heme is removed, rendering the empty heme binding pocket of rOM apo-b 5 more compact and less conformationally dynamic than that in bovine Mc (bMc) apo-b 5 . This may contribute to the stronger heme binding by OM apo-b 5 by reducing the entropic penalty associated with polypeptide folding. We also show that when bMc apo-b 5 unfolds it adopts a structure that is more compact and contains greater nonrandom secondary structure content than unfolded rOM apo-b 5 . We propose that a more robust ␤-sheet in Mc apo-b 5 s compensates for the absence of the hydrophobic packing interactions that stabilize the heme binding pocket in OM apo-b 5 s. are the products of different genes, and phylogenetic analysis has revealed that they are more closely related to one another than to the lone b 5 gene product in plants, insects, and fungi (Guzov et al. 1996). On this basis, it was proposed that Mc and OM b 5 arose via duplication of an ancestral gene and subsequent functional divergence. Supporting this hypothesis is the recent discovery that plant b 5 , long known to reside in the ER membrane, is also distributed to the outer mitochondrial membrane (Zhao et al. 2003). Studies in our laboratory with recombinant proteins representing the soluble heme-binding domains of rat OM (rOM) b 5 (Rivera et al. 1992(Rivera et al. , 1994Silchenko et al. 2000) and human OM b 5 (Altuve et al. 2004) have revealed that functional divergence has led to substantial differences in the biophysical properties of the two mammalian b 5 isoforms. Nonetheless, the Mc and OM b 5 heme binding domains adopt essentially identical folds comprising two hydrophobic cores separated by a five-stranded ␤-sheet as shown in Figure 1 (Durley and Mathews 1996;Rodriguez-Maranon et al. 1996). Article published online ahead of print. Article and publication date are at

We describe detailed studies of peptide-sandwiched mesohemes PSMA and PSMW, which comprise two histidine (His)-containing peptides covalently attached to the propionate groups of iron mesoporphyrin II. Some of the energy produced by ligation of the His side chains to Fe in the PSMs is invested in inducing helical conformations in the peptides. Replacing an alanine residue in each peptide of PSMA with tryptophan (Trp) to give PSMW generates additional energy via Trp side chain-porphyrin interactions, which enhances the peptide helicity and stability of the His-ligated state. The structural change strengthened His-FeIII ligation to a greater extent than His-FeII ligation, leading to a 56-mV negative shift in the midpoint reduction potential at pH 8 (Em,8 value). This is intriguing because converting PSMA to PSMW decreased heme solvent exposure, which would normally be expected to stabilize FeII relative to FeIII. This and other results presented herein suggest that differences in stability may be at least as important as differences in porphyrin solvent exposure in governing redox potentials of heme protein variants having identical heme ligation motifs. Support for this possibility is provided by the results of studies from our laboratories comparing the microsomal and mitochondrial isoforms of mammalian cytochrome b5. Our studies of the PSMs also revealed that reduction of FeIII to FeII reversed the relative affinities of the first and second His ligands for Fe (K2III > K1III; K2II < K1II). We propose that this is a consequence of conformational mobility of the peptide components, coupled with the much greater ease with which FeII can be pulled from the mean plane of a porphyrin. An interesting consequence of this phenomenon, which we refer to as "dynamic strain", is that an exogenous ligand can compete with one of the His ligands in an FeII-PSM, a reaction accompanied by peptide helix unwinding. In this regard, the PSMs are better models of neuroglobin, CooA, and other six-coordinate ligand-sensing heme proteins than of stably bis(His)-ligated electron-transfer heme proteins such as cytochrome b5. Exclusive binding of exogenous ligands by the FeII form of PSMA led to positive shifts in its Em,8 value, which increases with increasing ligand strength. The possible relevance of this observation to the function of six-coordinate ligand-sensing heme proteins is discussed.