12530
Biochemistry 2006, 45, 12530-12538
Evaluating the Roles of the Heme a Side Chains in Cytochrome c Oxidase Using Designed Heme Proteins† Jinyou Zhuang,‡ Amit R. Reddi,‡ Zhihong Wang,§,| Behzad Khodaverdian,§ Eric L. Hegg,*,§,| and Brian R. Gibney*,‡ Department of Chemistry, Columbia UniVersity, 3000 Broadway, MC 3121, New York, New York 10027, and Department of Chemistry, UniVersity of Utah, 315 South 1400 East, Salt Lake City, Utah 84112 ReceiVed March 21, 2006; ReVised Manuscript ReceiVed August 7, 2006
ABSTRACT:
Heme a is a redox cofactor unique to cytochrome c oxidases and vital to aerobic respiration. Heme a differs from the more common heme b by two chemical modifications, the C-8 formyl group and the C-2 hydroxyethylfarnesyl group. The effects of these porphyrin substituents on ferric and ferrous heme binding and electrochemistry were evaluated in a designed heme protein maquette. The maquette scaffold chosen, [∆7-H3m]2, is a four-R-helix bundle that contains two bis(3-methyl-L-histidine) heme binding sites with known absolute ferric and ferrous heme b affinities. Hemes b, o, o+16, and heme a, those involved in the biosynthesis of heme a, were incorporated into the bis(3-methyl-L-histidine) heme binding sites in [∆7-H3m]2. Spectroscopic analyses indicate that 2 equiv of each heme binds to [∆7H3m]2, as designed. Equilibrium binding studies of the hemes with the maquette demonstrate the tight affinity for hemes containing the C-2 hydroxyethylfarnesyl group in both the ferric and ferrous forms. Coupled with the measured equilibrium midpoint potentials, the data indicate that the hydroxyethylfarnesyl group stabilizes the binding of both ferrous and ferric heme by at least 6.3 kcal/mol via hydrophobic interactions. The data also demonstrate that the incorporation of the C-8 formyl substituent in heme a results in a 179 mV, or 4.1 kcal/mol, positive shift in the heme reduction potential relative to heme o due to the destabilization of ferric heme binding relative to ferrous heme binding. The two substituents appear to counterbalance each other to provide for tighter heme a affinity relative to heme b in both the ferrous and ferric forms by at least 6.3 and 2.1 kcal/mol, respectively. These results also provide a rationale for the reaction sequence observed in the biosynthesis of heme a.
The integral membrane protein complex cytochrome c oxidase (CcO)1 (E.C. 1.9.3.1) is the terminal enzyme in the energy-transducing, electron-transfer chain in all plants, animals, aerobic yeasts, and some bacteria (1-5). A vital component of aerobic metabolism, CcO catalyzes the four electron-four proton reduction of dioxygen to water along with the concomitant pumping of as many as four protons (eight charge equivalents) across the inner mitochondrial † This work was supported by an American Heart Association grant to B.R.G. (0455900T) and a National Institutes of Health grant to E.L.H. (GM 66236). A.R.R. acknowledges receipt of a National Science Foundation GK-12 Fellowship (DGE-02-31875). * Corresponding author. E-mail:
[email protected]. Phone: (212) 854-6346. Fax: (212) 932-1289. For questions concerning heme isolation, synthesis, purification, or characterization, contact
[email protected]. ‡ Columbia University. § University of Utah. | Current address: Department of Biochemistry and Molecular Biology, Michigan State University, 510 Biochemistry Building, East Lansing, MI 48824-1319. 1 Abbreviations: CcO, cytochrome c oxidase; H3m, 3-methyl-Lhistidine; TFA, trifluoroacetic acid; ESI/MS, electrospray ionization mass spectrometry; Fmoc, 9-fluorenylmethoxycarbonyl; tBoc, tertbutoxycarbonyl; PAL-PEG-PS, peptide amide linker-polyethylene glycol-polystyrene; HATU, O-(7-azabenzotriazol-1-yl)tetramethyluronium; OtBu, tert-butyl ester; Trt, trityl; Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; HPLC, high-performance liquid chromatography; Em8, midpoint reduction potential at pH 8.0; SHE, standard hydrogen electrode; Fe(DADPIX), iron diacetyldeuteroporphyrin IX.
membrane (6-10). Cytochrome c oxidases contain two redox centers, a binuclear CuA site and a bis-histidine-ligated heme a, that serve as an electron-transfer chain between watersoluble cytochrome c and the active site of CcO (1-3, 11). The electrons delivered by cytochrome c are shuttled to the buried heterobimetallic catalytic center composed of a highspin, monohistidine coordinated heme a3 and the CuB ligated by three histidines as shown in Figure 1. The heme a prosthetic group is unique to terminal oxidases and is biosynthetically derived from heme b (protoheme) using two enzymes as shown in Figure 2. First, heme b is transformed into heme o by converting the vinyl group at C-2 into a hydroxyethylfarnesyl group by heme o synthase (12-15). Second, heme a synthase oxidizes the methyl group at C-8 on heme o to a formyl group to produce heme a (15-19). An alcohol intermediate, heme o+16, has been isolated in the conversion of heme o into heme a (19, 20). Despite the importance of the heme a prosthetic group, relatively little is known about the role of the C-2 hydroxyethylfarnesyl and C-8 formyl side chains of heme a in the electron transfer and dioxygen reduction activity of CcO (15). Our approach to clarifying the structure-function relationships in natural heme proteins such as CcO is to evaluate the equilibrium thermodynamics of heme affinity and electrochemistry in simplified de novo designed heme proteins, or heme protein maquettes (21). By measuring the
10.1021/bi060565t CCC: $33.50 © 2006 American Chemical Society Published on Web 09/21/2006
Side Chain Structure-Function Studies of Heme a
Biochemistry, Vol. 45, No. 41, 2006 12531 protein interior counterbalances the weakened ferric heme affinity for the axial ligands due to the electronic effect of the formyl group at C-8. MATERIALS AND METHODS
FIGURE 1: Active site structure of bovine cytochrome c oxidase showing the heme a, heme a3, and CuB redox centers. The iron ions are represented by red spheres, and the copper ion is represented by a green sphere. The figure was prepared with MOLMOL (54).
absolute thermodynamic affinity of designed proteins for ferric and ferrous hemes along with the related electrochemistry, we are elucidating the fundamental factors that govern the reduction potentials of hemes in biology (22). We have designed a stably-folded four-R-helix bundle, [∆7-His]2 or [∆7-H10I14I21]2, and measured the absolute ferric and ferrous heme b affinities for its two bis-His heme binding sites (22). The heme b-[∆7-His]2 maquette displays spectroscopic properties similar to those of natural bis-His coordinated b-type cytochromes, e.g., cyt b5. Using a variety of non-natural amino acid ligands in a heme protein maquette, we have explored the effects of ligand basicity on ferric and ferrous heme affinities and found that the more basic 3-methyl-L-histidine had a slightly tighter heme affinity than L-histidine (23, 24). Additionally, we have demonstrated that electron-withdrawing substitutents on porphyrins elevate heme reduction potentials by destabilizing the ferric heme with no alteration in ferrous heme affinity for a designed bis-imidazole protein (25). In this paper, we report the spectroscopic, thermodynamic, and electrochemical consequences of the C-2 farnesyl and C-8 formyl groups of heme a in a designed heme protein maquette scaffold. The [∆7-H3m]2 maquette scaffold, which contains two bis(3-methyl-L-histidine) heme binding sites, was chosen for this study because its absolute affinity for ferric and ferrous heme b has been determined and it binds heme b more tightly than does the histidine maquette, [∆7His]2 (25). In order to separate the contributions of the C-2 farnesyl group from those of the C-8 formyl moiety, the series of hemes involved in the natural biosynthesis of heme a (heme b, heme o, heme o+16, and heme a) have been incorporated into this maquette scaffold. The affinity of the maquette scaffold for each heme in both the ferric and ferrous forms was measured and correlated with the related electrochemistry. On the basis of the thermodynamic relationship between the equilibrium midpoint reduction potential and the dissociation constants for the ferric and ferrous hemes, we demonstrate that one role of the C-2 hydroxyethylfarnesyl chain is to stabilize the binding of the heme macrocycle to the hydrophobic protein core by at least 6.3 kcal/mol in both the ferric and ferrous oxidation states. We also show that one role of the C-8 formyl group is to raise the midpoint reduction potential of the bound iron by 179 mV, or 4.1 kcal/ mol. The enhanced affinity due to the farnesyl tail for the
Materials. Isolation of heme a was performed as previously described (19). Heme b (ferriprotoporphyrin IX chloride) was purchased from Alfa Aesar. Fmoc-protected amino acids were acquired from Applied Biosystems (Framingham, MA) with the exception of Fmoc-L-H3m-OH, which was purchased from Bachem (King of Prussia, PA). All other chemicals were obtained from Fisher Scientific, VWR, or Sigma-Aldrich-Fluka unless otherwise noted. Expression of Heme o Synthase. The gene encoding for heme o synthase in Bacillus subtilis (ctaB) was cloned into Escherichia coli BL21(DE3) pLysS cells as previously described (20). Overnight cultures prepared from single colonies were used to inoculate (1:100 dilution) 500 mL of LB media containing 25 mg/L chloramphenicol and 50 mg/L ampicillin in 2 L indented flasks. Alternatively, overnight cultures were used to inoculate 15 L of LB media (1:50 dilution) in a 19 L Bellco Bioreactor System (O2 rate of 4 L/min). In both cases, cells were grown to an OD600 of 1.2 (measured on an HP 8453 diode array UV-visible spectrophotometer), induced with 75 mg/L isopropyl β-D-1-thiogalactopyranoside (IPTG) for 1-2 h, washed with a 0.25 M sucrose solution, and stored at -80 °C. Isolation and Purification of Heme o. To isolate heme o, the cells were thawed and resuspended in 10 mL of distilled water and 30 mL of a 5% HCl-acetone solution (per liter of culture). The cells were sonicated, vortexed vigorously (3 × 30 s), and then incubated at room temperature for 10 min. The mixture was then diluted with 10 mL of distilled water (per liter of culture), mixed, and clarified by centrifugation at 4400g for 15 min. Heme analysis of the supernatant indicated a heme b:heme o ratio of approximately 30:70 as determined by HPLC (20). To purify the heme o, the supernatant was loaded in 5 mL portions onto a Waters SepPak Vac C18 cartridge (12 cm3 g) that was preequilibrated with 15 mL of 25:75 CH3CN-H2O (0.1% trifluoroacetic acid). After loading, the cartridge was washed with an additional 15 mL of 25:75 CH3CN-H2O (0.1% TFA) followed by 80:20 CH3CN-H2O (0.1% TFA) until all of the heme b was eluted (approximately 25-50 mL/L of cell culture). Purified heme o was eluted from the column with DMSO (5 mL/L of cell culture), lyophilized, and stored at -80 °C. Chemical Reduction of Heme a to Heme o+16. The C-8 aldehyde group on heme a was anaerobically reduced to an alcohol under basic conditions using a procedure modified from Vanderkooi et al. (26). In short, HPLC-purified heme a (in 60% CH3CN-40% H2O-0.1% TFA) was mixed with an equal volume of 50 mM NaOH in 20% pyridine, capped, and sparged with N2. This solution was then incubated anaerobically with excess dithionite and NaBH4 for 20 min, followed by the addition of HCl to adjust the pH to 3. The reduced heme a was purified from the final solution by reversed-phase HPLC. The identity of the purified heme o+16 was confirmed by UV-visible spectroscopy and ESI/ MS (20), lyophilized, and stored at -80 °C. Chemical Synthesis of Peptides. General Procedure. The peptide was synthesized on a continuous flow Applied
12532 Biochemistry, Vol. 45, No. 41, 2006
Zhuang et al.
FIGURE 2: Chemical structures of each heme used in this study and their relationship to the heme a biosynthetic pathway.
Biosystems Pioneeer solid-phase peptide synthesizer using the Fmoc/tBu protection strategy with PAL-PEG-PS resin at 0.2 mmol scale (27). Single-coupling cycles of 30 min with HATU-activation chemistry were employed for all amino acids. The following side chain protecting groups were used: tBoc (Lys); OtBu (Glu); Trt (Cys); Pmc (Arg). After peptide assembly, the N-terminus was manually acetylated with acetic anhydride followed by thorough washing with DMF, MeOH, and CH2Cl2. The peptide was cleaved from the resin and simultaneously deprotected using 90:8:2 (v/v/ v) trifluoroacetic acid-ethanedithiol-water for 3 h. The crude peptide was precipitated and washed with cold ether, followed by dissolution in water [0.1% (v/v) TFA] and lyophilization. The peptide was purified by reversed-phase C18 HPLC using aqueous acetonitrile gradients containing 0.1% (v/v) TFA. The N-terminal cysteine residues of the purified peptide were air-oxidized to the symmetric disulfides in 100 mM ammonium carbonate buffer, pH 9.5 (4-7 h), which was followed by analytical HPLC. The resulting diR-helical disulfide-bridged peptide was identified with matrix-assisted laser desorption mass spectrometry (MALDIMS). UV-Visible Spectroscopy. UV-visible spectra were recorded on Varian Cary 100 and Perkin-Elmer Lambda 25 spectrophotometers using quartz cells of 1.0 and 10 cm path length. Peptide concentrations were determined spectrophotometrically using 280 of 5600 M-1 cm-1 per helix (28). Heme Affinity Studies. Ferric Heme. Freshly prepared DMSO solutions of heme b, heme o, heme o+16, or heme a were added in 0.1 equiv aliquots to peptide solutions (20 mM KPi, 100 mM KCl, pH 8.0). The concentration of the stock solutions was determined using the pyridine-hemochrome method which involves formation of the bis-pyridine ferrous heme complex in basic solution (29). Heme b equilibrated within 5 min while hemes o, o+16, and a required 2 h of equilibration between additions. The dissociation constant values were obtained from fitting the absorbance at λmaxox plotted against [heme]/[four-helix bundle] with a nonlinear least-squares routine in Kaliedagraph (Synergy Software, Reading, PA).
Absmeas ) Abs0 + bl[HP] + f l([Ht] - [HP]) [HP] )
[
(1)
]
Kd + [Pt] + [Ht] - x(-Kd - [Pt] - [Ht])2 - (4[Pt][Ht]) 2 (2)
The absorbance measured, Absmeas, is a sum of the initial
absorbance, Abs0, and the absorbance due to bound heme, bl[HP], and free heme, fl([Ht] - [HP]), where [Ht] and [HP] are the concentrations of total heme added and heme protein complex (bound heme), respectively, b and f are the extinction coefficients of bound and free heme, respectively, l is the path length of the cuvette, Kd is the conditional equilibrium dissociation constant of the heme-peptide complex, and [Pt] is the total peptide concentration (30). The values of the bound extinction coefficients were determined by converting each heme protein into the corresponding bispyridine hemochrome complex. After the spectrum of the oxidized and reduced heme protein was recorded in a 1.0 cm cuvette, the sample was diluted into a 0.1 N solution of NaOH containing 20% (v/v) pyridine and sodium dithionite. The spectrum of the pyridine hemochrome thus produced was recorded and the concentration of the heme was determined using the reported extinction coefficients of the heme b, heme o, and heme a pyridine hemochromes (29). These heme concentrations and the dilution factor between the original heme protein maquette sample and the pyridine hemochrome were used to determine the extinction coefficient of each bound heme, b, for use in the data analysis. Since the pyridine hemochrome of heme o+16 is not known, the values for heme o were used on the basis of the spectra similarity of their maquette complexes. Titrations of each heme into aqueous buffer without peptide present were performed to determine their free heme extinction coefficients, f. The slope of the resulting absorbance vs concentration curves yields the extinction coefficients of the unbound hemes which were used in the nonlinear leastsquares fitting analysis of heme affinity. Heme Affinity Studies. Ferrous Heme. Freshly prepared DMSO solutions of heme b and heme o were added in 0.06 equiv aliquots through a septum into anaerobic peptide solutions (20 mM KPi, 100 mM KCl, pH 8.0). Heme o was allowed to equilibrate for 2 h between additions. The samples were maintained at a reducing potential by the addition of sodium dithionite prior to the start of the titration. Due to the length of a typical heme o titration (78 h), the spectrophotometer was placed in an AtmosBag (Sigma-AldrichFluka, Milwaukee, WI) under a nitrogen atmosphere to ensure an anaerobic atmosphere. Initial attempts to measure the individual Kd1Fe(II) and Kd2Fe(II) values used 1.0 cm cuvettes, before 10 cm cuvettes were employed in an attempt to measure these tight values. The Kd values were obtained from fitting the absorbance at λmaxred plotted against [heme]/ [four-helix bundle] according to eq 1. Redox Potentiometry. Chemical redox titrations were performed in a 1.0 cm path length anaerobic cuvette equipped
Side Chain Structure-Function Studies of Heme a Scheme 1: Thermodynamic Analysis of Heme b-[∆7-H3m]2 Electrochemistry and Heme Affinity
with a platinum working and a calomel reference electrode at 22 °C (31). Ambient redox potentials (measured against the standard hydrogen electrode) were adjusted by addition of aliquots (
