Zn-myoglobin and Cytochrome b
5
A R T I C L E S
collected by vacuum filtration, and purified by silica gel column
chromatography. The desired product eluted as a red band with
methanol as eluent after the remaining starting material and byproducts
trometer equipped with a 5 mm triple resonance gradient probe using
128 × 512 complex points and spectral width of 365 × 8000 Hz in
the d
1
× d
2
dimensions. The 2D 15N-edited [ H, H]-NOESY-HSQC
1
1
were eluted with CH
evaporation gave 90 mg (60%) of the product. H NMR (500 MHz,
2
Cl
2
and ethyl acetate. Removal of the solvent by
spectrum with a mixing time of 150 ms was acquired using 128 × 512
complex points and spectral width of 6001 × 6001 Hz in the d × d
dimensions. The data were processed using Felix 97 (MSI, San Diego).
A sinebell window function was applied in both d and d dimensions.
1
1
2
DMSO): δ 3.04 (t, 4H), 3.60-3.74 (m, 12H), 4.32 (t, 4H), 6.90 (s,
1
3
2
1
1
H), 7.60 (s, 2H), 9.22 (d, 2H), 10.05-10.23 (m, 4H). C NMR: δ
2.11, 14.13, 22.93, 97.83, 100.57, 101.31, 130.25, 137.26, 141.09,
47.69, 148.19, 148.81, 174.89. MS (EI): 571.4.
1
2
The spectra were zero-filled to give a final matrix size of 256 × 1024.
Chemical shifts were referenced to the water peak at 4.64 ppm.
Electron-Transfer Pathway Analysis. The principles of ET pathway
analysis are based on the approximate method PATHWAY introduced
Synthesis of Zinc-deuteroporphyrin IX 15N-Diamide. The pro-
cedure was the same as that used to prepare zinc-deuteroporphyrin IX
1
5
21
diamide, except the aluminum amide reagent was prepared using NH
4
-
by Beratan and Onuchic et al. Electronic couplings in the PATHWAY
1
Cl. H NMR (500 MHz, DMSO): δ 3.04 (t, 4H), 3.60-3.74 (m, 12H),
model were computed as a product of decay factors associated with
covalent bonds (ꢀbond), nonbonded contacts (ꢀnb), and hydrogen bonds
(ꢀhb) between atoms on a path from the donor to the acceptor atom.
Electronic coupling maps were generated by calculating the electronic
tunneling matrix element from the redox cofactors in the protein to
4
.32 (t, 4H), 6.77-7.76 (q, 4H), 9.22 (d, 2H), 10.05-10.23 (m, 4H).
C NMR: δ 12.11, 14.13, 22.93, 97.83, 100.57, 101.31, 130.25, 137.26,
41.09, 147.69, 148.19, 148.81, 174.89. MS (EI): 573.3.
1
3
1
Preparation of Zinc-Substituted Mb’s. ZnMb was prepared with
11
22,23
the procedure we described previously. ZnMb(dme), ZnMb(diamide),
each atom on the protein surface.
1
5
and ZnMb( N-diamide) were prepared with a modified procedure
described here. First, apoMb was prepared using the method of Teale18
and dialyzed against 25 mM, pH 6.0 KPi buffer overnight. A 1.5-fold
molar excess of one of the zinc-porphyrins, either the ester or the amide,
Brownian Dynamic (BD) Simulation. BD simulations were per-
formed with the Macrodox program developed by Northrup and co-
workers at Tennessee Tech. The details of BD simulation of protein-
protein interaction have been described elsewhere.2 Briefly, the X-ray
crystallographic coordinates of ferri-Mb (1YMB) and cyt ferri-b5
(1CYO) were downloaded from the protein data bank, and the
coordinates for ZnMb and ZnMb(dme) were generated in WebLab
(MSN) by replacing the iron atom of the Mb coordinates with zinc
atom and adding two methyl groups to the propionates for ZnMb(dme).
4,25
3
was dissolved in a warm CH OH/DMSO (3:1 v/v) solution. After being
cooled to 4 °C, the porphyrin solution was added dropwise to the apoMb
solution with stirring at 4 °C, and the protein solution was kept in the
dark at 4 °C for 2 h. A second 1.5-fold molar excess of porphyrin was
then added in the same fashion, and the solution was kept at 4 °C for
another 2 h. The progress of the reconstitution process was followed
by monitoring the formation of the 414 nm absorption band of the
product in the UV-vis spectrum. In the reconstitution process, the total
volume of the organic solution added into the protein solution was kept
below 8% percent of the apo-Mb solution to prevent protein denatur-
ation. Following dialysis against phosphate buffer (pH 6.0, 25 mM)
overnight, the protein solution was concentrated and loaded onto a
Sephadex G-25 column to remove remaining free porphyrin. The protein
was further purified by HPLC with a TSK-based cation-exchange
column (Beckman, 21.5 mm × 15 cm, SP-5PW) and a 25 mM KPi
pH gradient (pH range, 6.0-12.0).
The coordinates for the tryptic cyt b were generated by deleting the
5
terminal residues flanking the tryptic fragment. In the program, charges
are assigned on the basis of the pK values estimated by Tanford-
a
Kirkwood calculation. On the basis of those assigned charges, a
Poisson-Boltzmann calculation is performed to determine the elec-
trostatic potential grid surrounding ZnMb. In the simulation, cyt b is
5
simply treated as an array of test charges in the field of ZnMb without
considering its internal low dielectric. The simulation starts with the
center of cyt b 70 Å away from the ZnMb potential grid. The Brownian
5
motion of cyt b in the field of ZnMb was simulated stochastically by
5
a series of small displacements governed by the Smoluchowski diffusion
equation with forces. A trajectory would be declared to be successful
Kinetics Measurements. Proteins were exchanged into working
buffer using Centricon microconcentrators (Amicon). Anaerobicity was
achieved by bubbling nitrogen through buffer solution in a sealed
cuvette for an hour, and gently purging the protein stock solution with
nitrogen for 20 min prior to addition to the sample cuvette. Kinetic
measurements were performed on two previously described laser flash
photolysis apparatus. The majority employed an apparatus with a time
constant of 15 µs and a continuous tungsten lamp as probe light
if the distance between any meso carbon (C
prescribed distance from any C of the partner heme at any time before
cyt b passes outside an escape radius of 200 Å. For each successful
trajectory, the position of cyt b at the instant of contact with the surface
M
) of one heme reaches a
M
5
5
of ZnMb is recorded, although the trajectories were not truncated until
the outer 200 Å sphere was reached.26 For ZnMb, the simulation with
10 000 trajectories under the condition of pH 7.0, 18 mM ionic strength
11
source; the most rapid reactions were monitored on an apparatus with
2
(10 mM KPi buffer) took 3 h of CPU time (SGI O ), while the
a time constant of a few nanoseconds and a xenon flash lamp as probe
light source.
simulation took 20 h for Zn(dme)Mb. A simulation generated a large
set of complexes, and they are represented here as a so-called docking
1
9,20
Isothermal Titration Calorimetry. Isothermal titration calorimetry
5
profile in which the centers of mass of cyt b in each complex are
(ITC) experiments were performed on an OMEGA titration microcalo-
represented as yellow dots surrounding ZnMb or ZnMb(dme).
rimeter from MicroCal, Inc. The proteins were exchanged into desired
buffer by dialysis against 4 L of 10 mM, pH 6.0 KPi buffer overnight
at 4 °C, and degassed using a vacuum pump. The titration was carried
Results
ET Kinetics. The three zinc deuteroporphyrin-substituted
ZnMb’s (Figure 1) have virtually identical UV-vis spectra, with
5
out by adding a ZnMb or ZnMb(dme) solution into a cyt b solution
(
∼0.3 mM) at 20 °C. The baseline was corrected by subtracting the
data from the titration of protein solutions into buffer. The final data
were analyzed using the program ORIGIN (MicroCal, Inc.) incorporat-
ing equations for one-site binding.
(
21) Beratan, D. N.; Onuchic, J. N.; Winkler, J. R.; Gray, H. B. Science 1992,
258, 1740-1741.
(22) Nocek, J. M.; Zhou, J. S.; De Forest, S.; Priyadarshy, S.; Beratan, D. N.;
Onuchic, J. N.; Hoffman, B. M. Chem. ReV. 1996, 96, 2459-2489.
NMR Experiment. The protein samples were prepared by dialysis
(
(
(
23) Roitberg, E. A.; Holden, M. J.; Mayhew, M. P.; Kurnikov, I. V.; Beratan,
1
15
against 4 L of 10 mM, pH 6.0 KPi overnight at 4 °C. The [ H, N]-
HSQC spectrum was acquired at 20 °C on a Varian INOVA spec-
D. N.; Vilker, V. L. J. Am. Chem. Soc. 1998, 120, 8927-8932.
24) Northrup, S. H.; Boles, J. O.; Reynolds, J. C. L. Science 1988, 241, 67-
70.
25) Northrup, S. H.; Thomasson, K. A.; Miller, C. M.; Barker, P. D.; Eltis, L.
D.; Guillemette, J. G.; Inglis, S. C.; Mauk, A. G. Biochemistry 1993, 32,
6613-6623.
(
18) Teale, F. W. J. Biochim. Biophys. Acta 1959, 35, 543.
19) Greenfield, S. R.; Svec, W. A.; Gosztola, D.; Wasielewski, M. R. J. Am.
Chem. Soc. 1996, 118, 6767-6777.
(
(26) Thus, the distance of closest approach for a trajectory with a particular
target distance could be less than that value.
(20) We thank Prof. M. Wasielewski for the use of this apparatus.
J. AM. CHEM. SOC.
9
VOL. 124, NO. 24, 2002 6851