The ci/bH moiety in the b6f complex studied by EPR: a pair of strongly interacting hemes

Abstract

X-band EPR features in the region of 90-150 mT have previously been attributed to heme ci of the b6 complex [Zhang H, Primak A, Bowman MK, Kramer DM, Cramer WA (2004) Biochemistry 43:16329-16336] and interpreted as arising from a high-spin species. However, the complexity of the observed spectrum is rather untypical for high-spin hemes. In this work, we show that addition of the inhibitor 2-n-nonyl-4-hydroxyquinoline N-oxide largely simplifies heme ci's EPR properties. The spectrum in the presence of 2-n-nonyl-4-hydroxyquinoline N-oxide is demonstrated to be caused by a simple S = 5/2, rhombic species split by magnetic dipolar interaction (A(xx )= 7.5 mT) with neighboring heme bH. The large spacing of lines in the uninhibited system, by contrast, cannot be rationalized solely on the basis of magnetic dipolar coupling but is likely to encompass strong contributions from exchange interactions. The role of the H2O/OH- molecule bridging heme ci's Fe atom and heme bH's propionate side chain in mediating these interactions is discussed.

Fig. 1.

EPR spectrum of the isolated b₆f complex from C. reinhardtii recorded at E_h = +200 mV. EPR settings were: temperature, 15 K; microwave power, 2.02 mW; microwave frequency, 9.42 GHz; modulation amplitude, 0.003231 mT; sweep time, 83.89 s. The spectral regions of the g_x/y signals of high-spin hemes (1), the g_z signals of cytochromes b and f (2), and the g_z,y,x signals of the Rieske center (3) are indicated. The amplitude in the low-field region has been multiplied by a factor of 5.

Fig. 2.

EPR spectra in the low-field region of (from top to bottom) membranes of C. reinhardtii, isolated cytochrome b₆f complex from C. reinhardtii, intact chloroplasts, and thylakoids from spinach for an untreated sample (A) and in the presence of NQNO (B). The amplitude of the signals was normalized to the amplitude of the Rieske spectrum after ascorbate reduction. EPR settings are as for Fig. 1. The amplitude of spectra from C. reinhardtii membranes is divided by 2 because they are contaminated by a strong iron signal at g = 9.35 and g = 4.2.

Fig. 3.

EPR spectra in the low-field region as recorded during a redox titration of the isolated b₆f complex from C. reinhardtii (30 μM). EPR settings are as for Fig. 1. (A) Shown are results with no inhibitor; ambient redox potentials from top to bottom are: +362, +198, +18, −104, and −232 mV. (B) Shown are results in the presence of 66 μM NQNO at ambient redox potentials of (from top to bottom) +414, +200, +15, −61, −97, −217, and −337 mV.

Fig. 4.

Evaluation of c_i 's redox behavior. (A) Redox-induced EPR difference spectra of the high-spin heme signals of the isolated b₆f complex from C. reinhardtii in the presence of 66 μM NQNO are shown; HP is +414/+200 mV; LP is −217/−337 mV. EPR settings are as for Fig. 1. (B) Signal amplitudes are plotted versus ambient redox potential: ■, g_y signal at 5.5; □, difference of the amplitudes of the g_x signals at 6.5 and 6.1. Two n = 1 Nernst curves were fitted to each set of data points. Asterisks indicate signal amplitude of the g_y signal at 5.32 from the titration without inhibitor versus ambient redox potential.

Fig. 5.

Orientation of c_i 's g tensor in the presence of NQNO. (A) EPR spectra were recorded on partially ordered multilayers of chloroplast membranes from spinach in the presence of NQNO. The angle between the magnetic field and the membrane plane is indicated for each spectrum. EPR settings are as for Fig. 1 except for temperature (6 K) and microwave power (63.8 mW). (B) Polar plots of signal amplitudes are shown.

Fig. 6.

Difference spectra of HP and LP components. Redox-induced EPR difference spectra of the high-spin heme signals of the isolated b₆f complex from C. reinhardtii are shown: HP, +362/+18 mV; LP, +18/−104 mV. EPR settings are as for Fig. 1.

Fig. 7.

g-tensor orientation with respect to the 3D structure. (Left) Structure of the c_i/b_H pair. The oxygen atom bridging heme c_i's central Fe atom to a propionate side-chain oxygen of heme b_H (Protein Data Bank ID code ) is highlighted. Bold arrows indicate the orientations of b_H's g_z and c_i's g_x directions. (Right) Schematic representation of the mutual orientations of the c_i/b_H pair and their maximal g values as seen from the n-side of the membrane.