Fuentes de información

The Dutton lab maquettes known as “BT6” and “BT6+” were utilized for all experiments in this chapter. Figure 2.1 illustrates the topology of the proteins. Figure 2.2 shows the sequences and predicted surface charges for BT6 and BT6+, along with the surface charge map for bovine cytochrome c [9]. These designs were originally published in [3].

BT6 a single-chain, water-soluble, 4-α-helical bundle with two bis-his sites for binding b-type or other iron porphyrins. The outward-facing portions of the helices are rich in glutamates and other charged residues, resulting in an expected overall charge of -16 at neutral pH. Both hemes bind tightly, with measured Kd values of < 5 nM for each heme. When 2 hemes are bound

each has a midpoint potential of -290 mV [3]. In lieu of hemes, the bis-His sites of BT6 will also bind two molar equivalents of the synthetic porphyrin iron diacetyldeuteroporphyrin IX, or Fe DADPIX (structure shown in Figure 1.2). The two electron-withdrawing acetyl substituents of DADPIX raise the Em to -

140 mV, while maintaining Kd < 100 nM

[10].

As in natural globins and some other maquettes, reduced iron porphyins in BT6 will readily bind diatomic gaseous

ligands including O2, nitric oxide, and carbon monoxide (CO) [3, 11, 12]. Bound CO stabilizes

the reduced heme state, rendering it unable to transfer electrons to potential redox partners such as cyt c. However, electronic excitation from a visible photon will release the bound CO, enabling the maquette to reduce an electron acceptor. Natural globins and some (but not all) other heme proteins share this property for CO[13] and other diatomic ligands [14]. Britton Chance employed flash photolysis to study ET kinetics in cytochrome c oxidase[15]. Most of this chapter’s experiments rely upon this established photolysis technique to initiate ET reactions. The photolysis data presented here employ CO bound to BT6 / heme, although BT6 / DADPIX

Figure 2.2. BT6 and BT6+ sequences. Basic and acidic residues are shown in blue and red, and heme- ligating histidines are green. Surface charge estimates

are shown for each maquette and for bovine cyt c,

generated using APBS[2].

BT6 (Em = -290 mV)

GEIWKQHEDALQKFEEALNQFEDLKQL GGSGSGSGG!

EIWKQHEDALQKFEEALNQFEDLKQL GGSGSGSGG!

EIWKQHEDALQKFEEALNQFEDLKQL GGSGSGSGG!

EIWKQHEDALQKFEEALNQFEDLKQL! BT6+ (Em = -150 m) GEIKRQHEDALRKFEEALKRFEDKKQK GGSGKGSGG! EIWKRHEDALRKFEEALKRFEDKKQK GGSGKGSGG! EIWKRHEDALRKFEEALKRFEDKKQK GGSGKGSGG! EIKRQHEDALRKFEEALKRFEDKKQK! Bovine Cyt c (Em = +250 mV)!

Figure 2. Sequences for molecules (1) and (2). Residues with positive and negative charges at pH 7.9 are shown in blue and red, and heme-ligating histidines are green. Electrostatic surface estimates are shown for each maquette and for bovine cyt c are were generated using APBS [APBS Citation]

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will also bind and photolyze a CO ligand. Figure 2.3 shows the UV-Vis spectra for the reduced, oxidized, and CO-bound species discussed in this chapter.

The BT6 scaffold has proven remarkably tolerant to mutation, binding a range of cofactors beyond iron porphyrins. One or both bis-His sites may be changed to single His to accommodate pentacoordinate zinc porphyrins or chlorins. Alternately, a cysteine may be introduced to bind a cytochrome P450-like heme or to provide a covalent flavin attachment[3].

Preliminary evidence shows BT6 can also bind more exotic ligands such as the multi-metal nitrogenase cofactor FeMoCo[16]. The 9-residue glycine / serine loops can be extended or shortened with minimal functional impact to the protein[3, 17].

Importantly for this work, the solvent-facing glutamates may also be changed to other hydrophilic, helix- forming residues. This opens the possibility to examine how surface charge impacts the maquette’s interaction with redox partners. Bovine cyt c contains a conspicuous patch of basic residues around its heme, yielding an overall charge of +7 units at physiological pH. This electrostatic feature influences its approach and orientation with physiological redox partners cytochrome bc1[18] and cytochrome

c oxidase (CcO) [19]. We expect a maquette’s charge to similarly impact its redox interaction with cyt c. BT6 is strongly negative, and thus charge complimentary with cyt c this should produce a fast interprotein ET rate. Likewise, we would expect slower ET from a positive maquette.

The variant known as “BT6+” replaces many of the glutamates with lysines, and the overall impact is to raise the total predicted charge from -16 to -11 at neutral pH. The

Figure 2.3. BT6 and cyt c spectra. UV-Vis spectra for redox-active cofactors examined in this chapter, measured on Varian spectrophotometer. Green trace is reduced BT6 heme bound to CO. BT6 spectra are molar extinction coefficients for a single heme (maquette has capacity to bind two hemes).

0 0.5 1 1.5 2 400 450 500 550 600 650 700 Cyt c (ox) Cyt c (red) BT6 Heme (ox) BT6 Heme (red) BT6 Heme (CO) BT6 DADPIX (ox) BT6 DADPIX (red)

ε

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preponderance of positive charge in BT6+ raises the heme Em to -150 mV[3]. The Em increase of

+140 mV, despite a large electrostatic change of 27 units, is relatively modest compared with similar large-scale charge changes in natural proteins. [20]. BT6+ binds both hemes with a reasonably tight 365 nM Kd [3].

Finally, it should be noted that although BT6 and BT6+ both exhibit strong charge monopoles, they were not designed as dipoles. Chapter 4 examines other maquettes with intentionally designed dipole moments.