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

Abstract

Microperoxidases were initially isolated as peptide fragments containing covalently bound heme and are derived from naturally occurring c-type cytochromes. They are not only used as model compounds but also have potential applications as biosensors, electron carriers, photoreceptors, microzymes, and drugs. In a systematic attempt to define the minimal requirements for covalent attachment of hemes to c-type cytochromes, we have succeeded to produce artificial microperoxidases with peptide sequences that do not occur naturally and can be manipulated. The in vivo production of these microperoxidases requires targeting of the peptide to the bacterial periplasm, proteolytic processing of the signal peptide, and covalent attachment of heme to the signature motif CXXCH by the cytochrome c maturation proteins CcmA-H. The peptides that bind heme carry a C-terminal histidine tag, presumably to stabilize the heme peptide. We present a heme cassette that is the basis for the de novo design of functional hemoproteins.

Fig. 3.

Length-yield correlation of purified microperoxidases. (A) Dithionite-reduced minus-air-oxidized difference spectra are shown. The y axis is expanded from 450 to 650 nm. For better resolution, the ΔA of MP370 and MP312 were magnified 3- and 10-fold, respectively. hh. cyt. c, horse heart cytochrome c. (B) Yields of heme peptides after purification over nickel nitrilotriacetic acid calculated from ΔA_{550–536 nm} of the difference spectra. The value of MP86 was set to 100%. (C) Correlation of length and yield of heme peptides. The y axis corresponds to that of B.

Fig. 1.

Biochemical and biophysical characteristics of MP86. (A) Heme stain of samples containing 1 nmol of heme peptide. Molecular weights of marker proteins and of the peptides are indicated on the left. (B) Optical spectra of MP86. Air-oxidized (ox.), reduced (5 mM dithionite; red.), and difference (dif.) spectra are shown in the presence (solid lines) and absence (dashed lines) of 300 mM imidazole. (Inset) The α/β region at higher magnification. (C) Results of MS. (D) Optical spectra for potentiometric titrations. (E) Determination of mid point potential with the Nernst equation. Curves from reductive titrations (▵) and oxidative titrations (□) as well as theoretical curves for n = 1 (one electron transfer) and E⁰ =–195 (solid line), –180 (dashed line), and –210 mV (dashed line) are shown.

Fig. 2.

Heme stains of purified microperoxidases. Equal volumes (10 μl) of purified peptides containing amounts of heme indicated in Fig. 3B were loaded in each lane. Molecular masses of marker proteins and of MP86 are indicated on the left.