Full assignment of heme redox potentials of cytochrome c₃ of D. vulgaris Miyazaki F by ¹H‐NMR

Abstract

Site-specific heme assignment of the 'H-NMR spectrum of cytochrome c, of D. vulgaris Miyazaki F, a tetraheme protein, was established. The major reduction of the heme turned out to take place in the order of hemes I, III, IV and II (numbering in the crystal struceure). The hemes with the smallest and greatest solvent accessibility were reduced at the highest and lowest potentials on average, respectively. A cooperative interhems inteeaction was attributed to a pair of the closest hemes. namely, hemes III and IV. This assignment can provide the physicochemical basis for the elucidation of electron transfer of this protein. Cyeochrome c,; Tetraheme protein; Redox potentials of a tctraheme protein; 'H-NMR; Assignment of eedox potential

Characterization of the structure and redox behaviour of cytochrome c3 from Desulfovibrio baculatus by 1H-nuclear-magnetic-resonance spectroscopy

“…The two phenylalanine patterns detected are due to the residues F72 or F88. Indeed, the resonances due to F34, the structurally conserved residue in all cytochromes c3 studied and which is in close contact with two haems [17], are shifted out of the aromatic region, as is also observed in the n.m.r. data obtained for the cytochromes c3 from D vulgaris [26] and D. gigas [27].…”

“…In particular, for these last two proteins, the intermolecular electron-exchange rate is low on the n.m.r. time scale [15,16], and the four haems have redox potentials of the same order of magnitude, with a maximum difference of 130 mV between the higherand lower-potential haems [15,17], while in the D. baculatus cytochromes the intermolecular exchange is intermediate to fast [18] and the higher-potential haem has a redox potential considerably higher (more than 150 mV) than the second-higher-potential one [23][24][25].…”

“…dynamic and kinetic, as well as structural, characteristics of the cytochromes C3, as the four low-spin haems act as intrinsic probes both in the reduced (diamagnetic) and especially in the oxidized (paramagnetic) and intermediate redox states [12][13][14][15][16][17][18][19].…”

NMR Studies of Cooperativity in the Tetrahaem Cytochrome c3 from Desulfovibrio vulgaris

“…then the overall pseudo-first-order rate constant for protonation is given by k,, = k, + (%) kOH (9) where Kw and K, are the dissociation constants of water and of the ionisable group on the protein, k, is the rate constant for protonation of the group in acid solution and koH is the rate constant for deprotonation of the group in basic solution [30,…”

“…cytochrome c, is a small (=14 m a ) , monomeric tetrahaem protein which exhibits cooperativity between the four haems and acidmase group(s): the haem redox potentials are pH dependent (redox-Bohr effect) and each haem redox potential is dependent on the oxidation state of the other three haems (redox interaction potentials) [4-61. Due to its small size and the fact that the haems are diamagnetic in the reduced state and paramagnetic in the oxidised one, NMR is particularly well suited to characterise this protein from the structural and thermodynamic point of view [4,[7][8][9][10][11][12][13][14][15].…”