Insight into Heme Protein Redox Potential Control and Functional Aspects of Six-Coordinate Ligand-Sensing Heme Proteins from Studies of Synthetic Heme Peptides

Abstract

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.

Heme Attachment Motif Mobility Tunes CytochromecRedox Potential

“…Acquisition of 2-D TOCSY or HSQC (of 15 N-labeled proteins) data commenced ~7 minutes after initiation of exchange. TOCSY spectra were collected (32 scans, 4096 × 256 points, 1.1-second recycle time) without solvent suppression at 8,14,19,25,30,35,40,95, and 315 hours after initiation of exchange. For Ht cyt c 552 , TOCSY spectra also were collected at 800, 1450, and 2650 hours after initiation of exchange.…”

“…Although there have been many studies of the effects of static polypeptide structure on heme-ligand interactions and on heme burial, the role of protein mobility has received less attention. Protein motions may indeed be important as they could influence metal-ligand interactions (15,18,19) and solvent exposure. † This work supported by National Institutes of Health Grant GM63170 (K.L.B.…”

Modulation of function in a minimalist heme-binding membrane protein

“…Water-soluble de novo designed heme proteins have long been used as models of more complicated, membraneembedded natural proteins that are involved in energy transduction [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. Several factors, including heme solvent exposure [9,10], stabilization of the peptide scaffold [2,6,15], and local electrostatic interactions [7], have been shown to modulate the redox potential and proton coupling of the cofactor in these models.…”

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

“…The goal of our study is to utilize MD simulations in an attempt to delineate the role of holoprotein dynamics in differentiating the stability and hemin release properties of OM and Mc b 5 , which may represent a source of their divergent redox potentials. 5,11,15,16 The results of such a study are also expected to contribute significantly to one of our major experimental research efforts-namely, the design of b 5 variants exhibiting properties such as improved thermal stability. 17 …”

“…13 In addition, recent studies in our laboratories have suggested that differences in stability may also represent part of nature's solution to differentiating the redox properties of the two b 5 isoforms. 15 …”