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The crystal structure of Ru(2,2′-bipyridine)2(imidazole)(His83)azurin (RuAz) has been determined to 2.3 Å resolution by X-ray crystallography. The spectroscopic and thermodynamic properties of both the native protein and [Ru(2,2′-bipyridine)2(imidazole)2]2+ are maintained in the modified protein. Dark-green RuAz crystals grown from PEG 4000, LiNO3, CuCl2 and Tris buffer are monoclinic, belong to the space group C2 and have cell parameters a = 100.6, b = 35.4, c = 74.7 Å and β = 106.5°. In addition, [Ru(2,2′-bipyridine)2(imidazole)2]SO4·10H2O was synthesized, crystallized and structurally characterized by X-ray crystallography. Red–brown crystals of this complex are monoclinic, space group P21/n, unit-cell parameters a = 13.230 (2), b = 18.197 (4), c = 16.126 (4) Å, β = 108.65 (2)°. Stereochemical parameters for the refinement of Ru(2,2′-bipyridine)2(imidazole)(His83) were taken from the atomic coordinates of [Ru(2,2′-bipyridine)2(imidazole)2]2+. The structure of RuAz confirms that His83 is the only site of chemical modification and that the native azurin structure is not perturbed significantly by the ruthenium label.
Supporting information
| Crystallographic Information File (CIF) Contains datablocks global, adb1 |
CCDC reference: 125242
PDB reference: ruthenium-modified azurin, 1bex
%TThe Ru atom position was found from the Patterson map and the
remaining heavy atom coordinates were obtained from subsequent
structure factor-Fourier calculations. H atoms were positioned by
calculation (C-H, 0.95 AA), and assigned
$B$ values 1.15 times the $U_{eq}$ of the
bonded C atom. H atoms of the
water molecules were ignored. The large displacement parameters for the
water oxygens may indicate partial occupancy. Two of the larger peaks
in the final difference map (1.18 and 1.01 eAA${-3}$) may also
indicate partial water molecules, although these peaks are within
1.9 AA\ of other waters. Thus, the exact water content of the crystal is
uncertain by $πm 2$ H$_2$O.
There is extensive hydrogen bonding among the sulfate oxygen atoms,
N7 and N8 of the imidazole groups and the water molecules, but in view
of the uncertainties in water content, a detailed description of this
network could not be developed. Atoms N7 and N8 of the
imidazole groups were assigned based on their heigths in the Fourier maps.
A least-squares calculation interchanging N7 and C22, and N8 and C25 gave
higher values of R-indices and goodness-of-fit, and gave displacement
parameters for those four atoms indicating that the original assignment
was correct.
Data collection: CAD-4 Software (Enraf-Nonius, 1989); cell refinement: CAD-4 Software (Enraf-Nonius, 1989); data reduction: CRYM (Duchamp, 1964); program(s) used to refine structure: CRYM (Duchamp, 1964); molecular graphics: ORTEPII (Johnson, 1976); software used to prepare material for publication: CRYM (Duchamp, 1964).
Crystal data top
C26H44N8O14RuS | Dx = 1.49 Mg m−3 |
Mr = 825.81 | Mo Kα radiation, λ = 0.7107 Å |
Monoclinic, P21/n | Cell parameters from 25 reflections |
a = 13.230 (2) Å | θ = 9–13° |
b = 18.197 (4) Å | µ = 0.54 mm−1 |
c = 16.126 (4) Å | T = 297 K |
β = 108.65 (2)° | Needle, red |
V = 3678.4 (15) Å3 | 0.43 × 0.10 × 0.07 mm |
Z = 4 | |
Data collection top
CAD-4 diffractometer | Rint = 0.031 |
Radiation source: Normal focus sealed tube | θmax = 20°, θmin = 1° |
Graphite monochromator | h = 0→11 |
Ω scans | k = −15→15 |
3430 measured reflections | l = −13→13 |
7815 independent reflections | 3 standard reflections every 150 minutes min |
7815 reflections with All | intensity decay: 13.8% |
Refinement top
Refinement on F2 | Primary atom site location: Patterson |
Least-squares matrix: full | Secondary atom site location: difference Fourier map |
R[F2 > 2σ(F2)] = 0.091 | Hydrogen site location: calculated |
wR(F2) = 0.17 | H-atom parameters not refined |
S = 3.53 | Calculated w = 1/σ2(Fo2) |
3429 reflections | (Δ/σ)max = 0.05 |
411 parameters | Δρmax = 1.24 e Å−3 |
0 restraints | Δρmin = −0.89 e Å−3 |
Crystal data top
C26H44N8O14RuS | V = 3678.4 (15) Å3 |
Mr = 825.81 | Z = 4 |
Monoclinic, P21/n | Mo Kα radiation |
a = 13.230 (2) Å | µ = 0.54 mm−1 |
b = 18.197 (4) Å | T = 297 K |
c = 16.126 (4) Å | 0.43 × 0.10 × 0.07 mm |
β = 108.65 (2)° | |
Data collection top
CAD-4 diffractometer | Rint = 0.031 |
3430 measured reflections | θmax = 20° |
7815 independent reflections | 3 standard reflections every 150 minutes min |
7815 reflections with All | intensity decay: 13.8% |
Refinement top
R[F2 > 2σ(F2)] = 0.091 | 0 restraints |
wR(F2) = 0.17 | H-atom parameters not refined |
S = 3.53 | Δρmax = 1.24 e Å−3 |
3429 reflections | Δρmin = −0.89 e Å−3 |
411 parameters | |
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) top | x | y | z | Uiso*/Ueq | |
Ru | 0.16626 (9) | 0.22528 (6) | 0.16175 (6) | 0.0429 (3) | |
N1 | 0.2801 (8) | 0.3057 (5) | 0.1919 (7) | 0.044 (3) | |
C1 | 0.3210 (12) | 0.3373 (9) | 0.1357 (9) | 0.068 (4) | |
C2 | 0.3927 (13) | 0.3958 (9) | 0.1592 (11) | 0.073 (5) | |
C3 | 0.4211 (13) | 0.4215 (8) | 0.2429 (12) | 0.080 (5) | |
C4 | 0.3794 (13) | 0.3896 (9) | 0.3008 (9) | 0.074 (5) | |
C5 | 0.3074 (11) | 0.3320 (7) | 0.2760 (9) | 0.046 (4) | |
C6 | 0.2587 (11) | 0.2955 (7) | 0.3308 (8) | 0.044 (4) | |
C7 | 0.2840 (12) | 0.3094 (8) | 0.4176 (10) | 0.075 (5) | |
C8 | 0.2358 (14) | 0.2695 (11) | 0.4680 (9) | 0.090 (6) | |
C9 | 0.1646 (12) | 0.2164 (9) | 0.4305 (9) | 0.071 (5) | |
C10 | 0.1402 (10) | 0.2045 (7) | 0.3415 (8) | 0.052 (4) | |
N2 | 0.1871 (8) | 0.2428 (6) | 0.2923 (6) | 0.040 (3) | |
N3 | 0.0540 (8) | 0.3046 (5) | 0.1121 (7) | 0.046 (3) | |
C11 | 0.0071 (13) | 0.3467 (8) | 0.1573 (9) | 0.063 (4) | |
C12 | −0.0689 (13) | 0.4005 (10) | 0.1189 (13) | 0.078 (6) | |
C13 | −0.0949 (15) | 0.4129 (10) | 0.0336 (16) | 0.105 (7) | |
C14 | −0.0460 (15) | 0.3710 (11) | −0.0154 (10) | 0.097 (7) | |
C15 | 0.0278 (12) | 0.3162 (8) | 0.0239 (11) | 0.060 (5) | |
C16 | 0.0792 (11) | 0.2676 (9) | −0.0214 (9) | 0.053 (4) | |
C17 | 0.0595 (13) | 0.2688 (10) | −0.1118 (10) | 0.083 (6) | |
C18 | 0.1082 (16) | 0.2228 (12) | −0.1509 (10) | 0.095 (7) | |
C19 | 0.1785 (14) | 0.1717 (10) | −0.0988 (11) | 0.084 (6) | |
C20 | 0.1976 (11) | 0.1711 (8) | −0.0066 (9) | 0.064 (4) | |
N4 | 0.1485 (8) | 0.2190 (7) | 0.0302 (6) | 0.045 (3) | |
N5 | 0.0447 (9) | 0.1464 (6) | 0.1370 (6) | 0.042 (3) | |
C21 | −0.0458 (13) | 0.1519 (8) | 0.1514 (8) | 0.051 (4) | |
N7 | −0.1096 (9) | 0.0925 (8) | 0.1217 (7) | 0.060 (4) | |
C22 | −0.0558 (14) | 0.0472 (8) | 0.0848 (9) | 0.064 (5) | |
C23 | 0.0383 (12) | 0.0791 (8) | 0.0941 (8) | 0.055 (4) | |
N6 | 0.2815 (9) | 0.1429 (6) | 0.2049 (6) | 0.047 (4) | |
C24 | 0.3771 (15) | 0.1394 (9) | 0.1958 (9) | 0.060 (5) | |
N8 | 0.4290 (10) | 0.0790 (9) | 0.2319 (9) | 0.074 (4) | |
C25 | 0.3653 (17) | 0.0412 (8) | 0.2662 (10) | 0.078 (6) | |
C26 | 0.2732 (12) | 0.0805 (9) | 0.2503 (9) | 0.061 (5) | |
S1 | 0.6914 (5) | 0.0109 (4) | 0.2035 (4) | 0.135 (2) | |
O1 | 0.6903 (11) | 0.0613 (10) | 0.1371 (9) | 0.226 (8) | |
O2 | 0.6387 (10) | 0.0474 (9) | 0.2540 (9) | 0.164 (6) | |
O3 | 0.8027 (11) | −0.0164 (8) | 0.2553 (10) | 0.159 (6) | |
O4 | 0.6212 (19) | −0.0501 (17) | 0.1580 (17) | 0.357 (15) | |
W1 | 0.9745 (9) | 0.0693 (6) | 0.3516 (8) | 0.116 (4) | |
W2 | 0.7327 (10) | 0.0829 (7) | 0.4297 (7) | 0.126 (4) | |
W3 | 0.8790 (11) | 0.1842 (8) | 0.4134 (8) | 0.151 (5)* | |
W4 | 0.1306 (13) | 0.0319 (8) | 0.5050 (10) | 0.184 (6)* | |
W5 | 0.1659 (14) | 0.1409 (9) | 0.6443 (10) | 0.190 (6)* | |
W6 | 0.9990 (15) | 0.2545 (10) | 0.5768 (11) | 0.220 (8)* | |
W7 | 0.3538 (14) | 0.2244 (10) | 0.7707 (11) | 0.210 (7)* | |
W8 | 0.6901 (15) | −0.0060 (11) | 0.9658 (11) | 0.227 (8)* | |
W9 | 0.500 (2) | 0.1086 (14) | 0.0306 (17) | 0.339 (13)* | |
W10 | 0.5488 (19) | 0.1017 (12) | 0.8662 (14) | 0.272 (10)* | |
H1 | 0.301 | 0.319 | 0.077 | 0.077* | |
H2 | 0.421 | 0.417 | 0.118 | 0.082* | |
H3 | 0.470 | 0.461 | 0.261 | 0.092* | |
H4 | 0.400 | 0.407 | 0.359 | 0.084* | |
H7 | 0.335 | 0.347 | 0.444 | 0.085* | |
H8 | 0.252 | 0.279 | 0.529 | 0.104* | |
H9 | 0.132 | 0.188 | 0.464 | 0.081* | |
H10 | 0.089 | 0.168 | 0.314 | 0.058* | |
H11 | 0.026 | 0.340 | 0.219 | 0.071* | |
H12 | −0.102 | 0.428 | 0.154 | 0.089* | |
H13 | −0.146 | 0.450 | 0.007 | 0.119* | |
H14 | −0.063 | 0.380 | −0.077 | 0.110* | |
H17 | 0.010 | 0.303 | −0.147 | 0.094* | |
H18 | 0.097 | 0.225 | −0.212 | 0.109* | |
H19 | 0.214 | 0.137 | −0.124 | 0.095* | |
H20 | 0.247 | 0.137 | 0.029 | 0.072* | |
H21 | −0.066 | 0.193 | 0.180 | 0.058* | |
HN7 | −0.179 | 0.085 | 0.126 | 0.070* | |
H22 | −0.080 | 0.002 | 0.057 | 0.073* | |
H23 | 0.093 | 0.059 | 0.075 | 0.062* | |
H24 | 0.405 | 0.176 | 0.167 | 0.068* | |
HN8 | 0.499 | 0.065 | 0.233 | 0.085* | |
H25 | 0.382 | −0.005 | 0.296 | 0.090* | |
H26 | 0.213 | 0.067 | 0.268 | 0.070* | |
Atomic displacement parameters (Å2) top | U11 | U22 | U33 | U12 | U13 | U23 |
Ru | 0.0431 (7) | 0.0529 (7) | 0.0317 (6) | −0.0065 (7) | 0.0106 (5) | −0.0030 (6) |
N1 | 0.038 (7) | 0.044 (7) | 0.051 (7) | −0.003 (5) | 0.017 (6) | 0.000 (6) |
C1 | 0.070 (11) | 0.087 (12) | 0.057 (10) | −0.021 (10) | 0.035 (9) | −0.002 (9) |
C2 | 0.076 (12) | 0.072 (12) | 0.081 (13) | −0.027 (10) | 0.040 (10) | 0.005 (10) |
C3 | 0.084 (13) | 0.079 (13) | 0.073 (12) | −0.028 (10) | 0.019 (11) | −0.006 (10) |
C4 | 0.094 (13) | 0.074 (12) | 0.051 (10) | −0.024 (11) | 0.017 (10) | −0.018 (9) |
C5 | 0.055 (10) | 0.045 (9) | 0.035 (9) | −0.011 (8) | 0.009 (8) | −0.014 (8) |
C6 | 0.056 (10) | 0.047 (10) | 0.031 (9) | −0.008 (8) | 0.019 (8) | −0.013 (7) |
C7 | 0.070 (11) | 0.091 (12) | 0.063 (11) | −0.045 (9) | 0.020 (9) | −0.023 (9) |
C8 | 0.097 (13) | 0.132 (15) | 0.045 (9) | −0.031 (13) | 0.027 (10) | −0.015 (11) |
C9 | 0.083 (12) | 0.094 (13) | 0.048 (10) | −0.028 (10) | 0.036 (9) | −0.006 (9) |
C10 | 0.037 (9) | 0.078 (11) | 0.036 (9) | −0.011 (7) | 0.005 (7) | −0.007 (7) |
N2 | 0.038 (7) | 0.052 (8) | 0.030 (6) | 0.001 (6) | 0.012 (6) | −0.001 (6) |
N3 | 0.048 (7) | 0.047 (8) | 0.045 (8) | −0.009 (6) | 0.019 (7) | −0.003 (6) |
C11 | 0.074 (12) | 0.056 (11) | 0.054 (10) | −0.002 (10) | 0.016 (9) | −0.009 (9) |
C12 | 0.063 (12) | 0.084 (14) | 0.082 (13) | 0.018 (10) | 0.016 (11) | −0.004 (11) |
C13 | 0.089 (15) | 0.096 (15) | 0.116 (18) | 0.043 (12) | 0.012 (14) | 0.011 (13) |
C14 | 0.078 (14) | 0.123 (17) | 0.068 (12) | 0.008 (13) | −0.011 (11) | 0.043 (12) |
C15 | 0.047 (10) | 0.072 (11) | 0.054 (11) | −0.007 (9) | 0.008 (9) | 0.011 (9) |
C16 | 0.056 (10) | 0.071 (10) | 0.034 (9) | −0.011 (10) | 0.016 (8) | 0.001 (9) |
C17 | 0.088 (13) | 0.108 (13) | 0.045 (11) | 0.014 (12) | 0.012 (9) | −0.002 (10) |
C18 | 0.102 (15) | 0.141 (17) | 0.036 (10) | −0.011 (14) | 0.013 (10) | 0.014 (12) |
C19 | 0.090 (14) | 0.129 (16) | 0.051 (12) | −0.034 (12) | 0.047 (10) | −0.034 (10) |
C20 | 0.063 (11) | 0.074 (11) | 0.059 (11) | −0.008 (9) | 0.025 (9) | −0.008 (8) |
N4 | 0.043 (7) | 0.064 (8) | 0.037 (7) | −0.005 (7) | 0.023 (6) | 0.001 (7) |
N5 | 0.040 (8) | 0.056 (9) | 0.035 (6) | −0.004 (6) | 0.017 (6) | 0.005 (6) |
C21 | 0.054 (11) | 0.061 (11) | 0.035 (8) | −0.002 (9) | 0.011 (8) | 0.000 (7) |
N7 | 0.047 (8) | 0.080 (10) | 0.049 (8) | −0.022 (8) | 0.009 (7) | 0.003 (7) |
C22 | 0.059 (12) | 0.065 (11) | 0.064 (11) | −0.022 (10) | 0.012 (10) | 0.001 (8) |
C23 | 0.066 (12) | 0.053 (10) | 0.043 (9) | 0.008 (9) | 0.015 (8) | −0.004 (8) |
N6 | 0.047 (9) | 0.050 (9) | 0.040 (7) | −0.001 (6) | 0.006 (6) | −0.006 (6) |
C24 | 0.055 (12) | 0.070 (13) | 0.055 (10) | 0.001 (10) | 0.016 (9) | −0.017 (9) |
N8 | 0.058 (9) | 0.090 (12) | 0.069 (9) | 0.015 (10) | 0.013 (8) | −0.022 (8) |
C25 | 0.081 (14) | 0.060 (12) | 0.076 (12) | 0.011 (12) | 0.003 (11) | 0.011 (9) |
C26 | 0.056 (11) | 0.064 (11) | 0.054 (10) | 0.002 (10) | 0.004 (8) | 0.001 (9) |
S1 | 0.086 (5) | 0.198 (7) | 0.120 (5) | −0.020 (5) | 0.030 (4) | −0.007 (5) |
O1 | 0.130 (13) | 0.35 (2) | 0.155 (13) | −0.062 (14) | −0.019 (10) | 0.204 (16) |
O2 | 0.091 (10) | 0.263 (18) | 0.137 (11) | 0.090 (11) | 0.036 (9) | −0.046 (11) |
O3 | 0.097 (11) | 0.181 (15) | 0.206 (16) | −0.009 (11) | 0.056 (11) | −0.029 (12) |
O4 | 0.21 (3) | 0.46 (4) | 0.35 (3) | −0.09 (3) | 0.00 (2) | −0.15 (3) |
W1 | 0.107 (9) | 0.110 (9) | 0.133 (10) | −0.003 (8) | 0.042 (8) | 0.011 (7) |
W2 | 0.123 (11) | 0.145 (11) | 0.111 (9) | 0.012 (9) | 0.040 (8) | −0.012 (8) |
Experimental details
Crystal data |
Chemical formula | C26H44N8O14RuS |
Mr | 825.81 |
Crystal system, space group | Monoclinic, P21/n |
Temperature (K) | 297 |
a, b, c (Å) | 13.230 (2), 18.197 (4), 16.126 (4) |
β (°) | 108.65 (2) |
V (Å3) | 3678.4 (15) |
Z | 4 |
Radiation type | Mo Kα |
µ (mm−1) | 0.54 |
Crystal size (mm) | 0.43 × 0.10 × 0.07 |
|
Data collection |
Diffractometer | CAD-4 diffractometer |
Absorption correction | – |
No. of measured, independent and observed (All) reflections | 3430, 7815, 7815 |
Rint | 0.031 |
θmax (°) | 20 |
(sin θ/λ)max (Å−1) | 0.481 |
|
Refinement |
R[F2 > 2σ(F2)], wR(F2), S | 0.091, 0.17, 3.53 |
No. of reflections | 3429 |
No. of parameters | 411 |
H-atom treatment | H-atom parameters not refined |
Δρmax, Δρmin (e Å−3) | 1.24, −0.89 |
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