Hydrogen exchange (HX) rates and midpoint potentials (E m ) of variants of cytochromes c from Pseudomonas aeruginosa (Pa cyt c 551 ) and Hydrogenobacter thermophilus (Ht cyt c 552 ) have been characterized toward developing an understanding of the impact of properties of the Cys-X-X-CysHis pentapeptide c-heme attachment motif (CXXCH) on heme redox potential. Despite structural conservation of the CXXCH motif, Ht cyt c 552 exhibits low protection from HX for amide protons within this motif relative to Pa cyt c 551 . Site-directed mutants have been prepared to determine the structural basis for and functional implications of these variations in HX behavior. The double mutant Ht-M13V/K22M displays suppressed HX within the CXXCH motif as well as decreased E m (by 81 mV), whereas the corresponding double mutant of Pa cyt c 551 (V13M/M22K) exhibits enhanced HX within the CXXCH pentapeptide and a modest increase in E m (by 30 mV). The changes in E m correlate with changes in axial His chemical shifts in the ferric proteins reflecting extent of histidinate character. Thus the mobility of the CXXCH pentapeptide is found to impact the His-Fe(III) interaction and therefore heme redox potential.Electron transfer reactions involving iron-protoporphyrin IX (heme) are central to fundamental biological processes such as respiration, redox catalysis, sensing, and signaling (1-5). A key parameter determining energetics and kinetics of electron transfer is the redox potential (1), thus, much emphasis has been placed on understanding the role of protein structure in tuning heme redox potential. Two fundamental features known to have a substantial influence on heme redox potentials are the nature of the ligands coordinated to the metal and the burial of the heme in the hydrophobic protein core. Nature alters the electron donating properties of the coordinating ligands through choice of ligands (6), modulating metal-ligand bond strength (6-12), varying coordination geometry (5), and hydrogen bonding to ligands (13)(14)(15). The encapsulation of the heme within a protein's interior also is significant for determining potential, as the hydrophobic environment favors the ferrous state over the ferric (7,8,10,16,17). 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.), a Fellowship from the Alfred P. Sloan Foundation (K.L.B.), and National Science Foundation Grant MCB-0546323 (S.J.E.).*To whom correspondence should be addressed: Department of Chemistry, University of Rochester, Rochester, NY 14627-0216. Telephone: (585) Here, we investigate the effects of structural fluctuations of the c-heme motif of cytochrome c (cyt c 1 ) on redox potential. The c-type heme is characterized by its covalen...

The direct electrochemical analysis of adsorbed redox active proteins has proven to be a powerful technique in biophysical chemistry, frequently making use of the electrode material pyrolytic “edge-plane” graphite. However many heme-bearing proteins such as cytochromes c have been also examined systematically at alkanethiol-modified gold surfaces, and previously we have reported the characterization of the redox properties of a series of bacterial cytochromes c in a side-by-side comparison of carbon and gold electrode materials. In our prior findings we reported an unanticipated, low potential (Em ~ -100 mV vs SHE) redox couple that could be analogously observed when a variety of monoheme cytochromes c are adsorbed onto carbon-based electrodes. Here we demonstrate that our prior phenomological data can be understood quantitatively in the loss of the methionine ligand of the heme iron, using the cytochrome c from Hydrogenbacter thermophilum as a model system. Through the comparison of wild-type protein with M61H and M61A mutants, in direct electrochemical analyses conducted as a function of temperature and exogenous ligand concentration, we are able to show that Met-ligated cytochromes c have a propensity to lose their Met ligand at graphite surfaces, and that energetics of this process (6.3 ± 1 kJ/mol) is similar the energies associated with “foldons” of known protein folding pathways.

Chronic traumatic encephalopathy (CTE) formerly known as dementia pugilistica is a long-term neurodegenerative disorder associated with repeated subconcussive head injuries in high-contact sports. We reviewed the existing literature on CTE and examined epidemiological trends, risk factors, and its temporal progression, and proposed the underlying pathophysiological mechanisms that may provide unique insights to clinicians with an in-depth understanding of the disease to aid in the diagnosis and prevention, and provide future perspectives for research via search of Medline and Cochrane databases as well as manual review of bibliographies from selected articles and monographs. The prevalence of CTE in recent years is on the rise and almost exclusively affects men, with pathologic signs characterized by progressive memory loss, behavioral changes, and violent tendencies with some patients demonstrating Parkinsonian-like symptoms and signs. Many patients with CTE die following suicide, accident, or complications of drug or alcohol use. Postmortem pathologic analysis is characterized by neurofibrillary tangles and Aβ plaques in 50 % of cases. Currently, there are no ante-mortem diagnostic criteria, but modern imaging techniques such as functional magnetic resonance (MR) imaging, MR spectroscopy, and diffusion tension imaging hold promise for delineating the future diagnostic criteria. Further long-term longitudinal studies are warranted to investigate risk factors that will enhance understanding of the disease progression and its pathogenesis.

Heme c is characterized by its covalent attachment to a polypeptide. The attachment is typically to a CXXCH motif in which the two Cys form thioether bonds with the heme, "X" can be any amino acid other than Cys, and the His serves as a heme axial ligand. Some cytochromes c, however, contain heme attachment motifs with three or four intervening residues in a CXCH or CXCH motif. Here, the impacts of these variations in the heme attachment motif on heme ruffling and electronic structure are investigated by spectroscopically characterizing CXCH and CXCH variants of Hydrogenobacter thermophilus cytochrome c. In addition, a novel CXCH variant is studied. H andC NMR, EPR, and resonance Raman spectra of the protein variants are analyzed to deduce the extent of ruffling using previously reported relationships between these spectral data and heme ruffling. In addition, the reduction potentials of these protein variants are measured using protein film voltammetry. The CXCH and CXCH variants are found to have enhanced heme ruffling and lower reduction potentials. Implications of these results for the use of these noncanonical motifs in nature, and for the engineering of novel heme peptide structures, are discussed.