The asymmetric distribution of charges on the surface of horse cytochrome c. Functional implications.

W H Koppenol,E Margoliash

doi:10.1016/s0021-9258(18)34740-9

Abstract

The electric potential field around native horse cytochrome c and 12 singly modified 4-carboxy-2,4-dinitrophenyl- (CDNP) lysine cytochromes c is asymmetric, mainly because of the inhomogeneous distribution of negative charges. Dipole moments of 325 and 308 debye, (1.08.10(-27) and 1.03.10(-27) coulomb.meter), respectively, were calculated for horse ferri- and ferrocytochrome c. The angle between the heme plane and the dipole vector of horse ferricytochrome c is 33 degrees and increases 1 degree upon reduction to the ferrous form. Dipole moments of the CDNP-lysine cytochromes c differ from that of native cytochrome c by as much as 140 debye in magnitude and 45 degrees in direction. It is proposed that its dipole moment causes cytochrome c to orient itself in the electric fields of its redox partners, and that the CDNP-lysine cytochromes c, which have different dipole moments, do not form a productive complex. Reorientation to the correct position for electron transfer increases the activation energy and lowers the rate of reaction. This model describes quantitatively the relative activities of those CDNP-lysine cytochromes c which are modified outside of the interaction domain and it allows correction of the activities of those modified inside the domain, on the front surface of the molecule, for the change in dipole moment. The interaction domain for the reaction with cytochrome c reductase includes in decreasing order of involvement lysines 13, 72, 86, 27, and 87. That for the reaction with cytochrome c oxidase is slightly smaller, with lysines 13, 72, 86, and 27. The cytochrome c peroxidase domain is the largest of all and is defined by lysines 72, 86, 13, 87, 27,, and 73. All refined interaction domains encompass the exposed heme edge and are to a large extent overlapping, indicating that electron transfer takes place at or close to this prosthetic group and that cytochrome c must move on the outer surface of the inner mitochondrial membrane during electron transport between reductase and oxidase. For a quantitative description of the electrostatic interaction of cytochrome c with other molecules, it is essential to take into account the totality of its charge configuration.

Highlights

The electric potential field around native horse cy- to, the exposed heme edge (1-12)
Directmeasurements by stopped flow show that dipole moment causes cytochrome c to orient itself in electron transfer between cytochrome c and its physiological the electric fields of its redox partners, and that the redox partners takes place at rates which are close to diffusion
All refined interaction domains encompass the exposed heme edge and are to a large extent overlapping, indicating that electron transfertakes place at or close tothis prosthetic group and that cytochrome c must move on the outer surface of the inner mitochondrial membrane during electron transport between reductase and oxidase

Summary

RESULTS

In the case of the CDNPderivatives,it is possible to observe a single charge, because the negative charge at the4-position of the phenyl group is assumed to be located in the aqueous phase at some distance from the protein surface and has a distinct effect on the distribution of potential surfaces This is evident, depicting a section through the electric potential field of CDNP-lysine 13 horse ferrocytochrome c, which, like all the chemically modified cytochromes c, shows the effect of the smaller net charge, f 6 e instead of +8 e. A similar approach to thereaction of modified cytochromes c with yeast cytochrome c peroxidase (5) appears to indicate that lysines 277 , and 87 are located in the interaction domain (Fig. 12).This figure should be interpreted with caution, since not enough points are available to identify with certainty the derivatives whose activities define the straight line.

DISCUSSION

Findings

FERRCKYTOCHROME c