Control of Cytochrome c Redox Potential:  Axial Ligation and Protein Environment Effects

Abstract

Axial iron ligation and protein encapsulation of the heme cofactor have been investigated as effectors of the reduction potential (E degrees ') of cytochrome c through direct electrochemistry experiments. Our approach was that of partitioning the E degrees ' changes resulting from binding of imidazole, 2-methyl-imidazole, ammonia, and azide to both cytochrome c and microperoxidase-11 (MP11), into the enthalpic and entropic contributions. N-Acetylmethionine binding to MP11 was also investigated. These ligands replace Met80 and a water molecule axially coordinated to the heme iron in cytochrome c and MP11, respectively. This factorization was achieved through variable temperature E degrees ' measurements. In this way, we have found that (i) the decrease in E degrees ' of cytochrome c due to Met80 substitution by a nitrogen-donor ligand is almost totally enthalpic in origin, as a result of the stronger electron donor properties of the exogenous ligand which selectively stabilize the ferric state; (ii) on the contrary, the binding of the same ligands and N-acetylmethionine to MP11 results in an enthalpic stabilization of the reduced state, whereas the entropic effect invariably decreases E degrees ' (the former effect prevails for the methionine ligand and the latter for the nitrogenous ligands). A comparison of the reduction thermodynamics of cytochrome c and the MP11 adducts offers insight on the effect of changing axial heme ligation and heme insertion into the folded polypeptide chain. Principally, we have found that the overall E degrees ' increase of approximately 400 mV, comparing MP11 and native cytochrome c, consists of two opposite enthalpic and entropic terms of approximately +680 and -280 mV, respectively. The enthalpic term includes contributions from both axial methionine binding (+300 mV) and protein encapsulation of the heme (+380 mV), whereas the entropic term is almost entirely manifest at the stage of axial ligand binding. Both terms are dominated by the effects of water exclusion from the heme environment.

Biosynthesis of artificial microperoxidases by exploiting the secretion and cytochrome c maturation apparatuses of Escherichia coli

“…The presence of a strong sixth ligand to the heme iron seems likely from the sharp ␣-and ␤-bands of the difference spectra, as expected for low-spin heme and from the midpoint potentials. Although acetylated MP-11 had a significant difference in midpoint potentials, depending on the presence (Ϫ189 mV) or absence (Ϫ134 mV) of imidazole (14), no such difference was found for MP86 and MP251. Furthermore, the midpoint potential of the H 6 -tagged heme peptides in the absence of imidazole is close to the midpoint potential of acetylated MP-11 in the presence of the strong iron ligand imidazole.…”

“…Hemebinding peptides, also referred to as microperoxidases (1), are used as biosensors (2,3), electron carriers (4-6), photoreceptors (7,8), molecules with specific electrochemical or catalytic properties (6, 9, 10), drugs (11), in biofuel cells (12), and as model compounds for understanding biological redox systems (5). Microperoxidases have been used to better characterize the structural and functional aspects of cytochromes (13), to obtain new, low molecular weight compounds able to mimic biologically active systems (14), and as molecular markers (15). So far, they have been synthesized chemically or by proteolytic digestion of naturally existing c-type cytochromes.…”

Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides

“…Electrochemical Properties of the Cyt c 1 -Im Complex-Binding of Im to horse heart cyt c results in a ϳ400-mV potential drop, to Ϫ135 mV (8,9), consistent with replacement of the native methionine ligand, yielding an imidazole-histidine ligated heme with properties similar to those of a typical bis-his heme. In both R. capsulatus and R. sphaeroides cyt c 1 , substitution of histidine for methionine as the sixth ligand led to photosynthetic incompetence, which was attributed to the lower redox potential of the mutant cyt c 1 (26,29,56).…”

“…In most cases, binding occurs preferentially to the ferric iron and causes substantial changes in the redox potential (8, 9) and spectral properties of the cytochrome (10 -12). These include cyanide, azide, fluoride, imidazole, pyridine, and nitric oxide (8,9,(13)(14)(15). For example, binding of imidazole (Im) to oxidized mitochondrial cyt c or bacterial cyt c 2 produces a blue shift in the Soret region as well as changes in a number of weak absorbance bands between 450 and 600 nm (10 -12).…”

“…In most cases, binding occurs preferentially to the ferric iron and causes substantial changes in the redox potential (8,9) and spectral properties of the cytochrome (10 -12). These include cyanide, azide, fluoride, imidazole, pyridine, and nitric oxide (8,9,(13)(14)(15).…”