Structures of ruthenium-modified Pseudomonas aeruginosa azurin and [Ru(2,2′-bipyridine)₂(imidazole)₂]SO₄·10H₂O

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.

S1. Refinement

%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.}

Computing details

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).

(adb1)

Crystal data

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) Å^3	0.43 × 0.10 × 0.07 mm
Z = 4

Data collection

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

Refinement on F^2	Primary atom site location: Patterson
Least-squares matrix: full	Secondary atom site location: difference Fourier map
R[F^2 > 2σ(F^2)] = 0.091	Hydrogen site location: calculated
wR(F^2) = 0.17	H-atom parameters not refined
S = 3.53	Calculated w = 1/σ^2(Fo^2)
3429 reflections	(Δ/σ)max = 0.05
411 parameters	Δρmax = 1.24 e Å^−3
0 restraints	Δρmin = −0.89 e Å^−3

Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Ų)

	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 (Ų)

	U^11	U^22	U^33	U^12	U^13	U^23
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)