Cytochrome bo 



565 



cytochrome b^, is easy to distinguish from that of cytochrome b of the bakers' yeast 

 cells. The former is illustrated by Fig. 1 which shows that the <xy and ao-bands of 

 cytochrome b^ He at 558 and 553-4 m/t. In the low temperature spectrum of yeast cells 

 (Chance, this volume, p. 233) the cytochrome b of the respiratory chain has an a peak 

 at a longer wavelength, about 560 m//, and no ao peak. These two curves clearly 

 distinguish the two cytochromes and show that cytochrome /?., is not involved to a 

 measurable extent in the respiratory chain of yeast cells. 



The Kinetics of Reactions Catalysed by Cytochrome \ 

 The Kinetics of Reduction of Cytoclirome b^ 



By B. Chance (Philadelphia) 

 Chance : In spite of the great progress that has been made in the preparation of types of 

 lactate dehydrogenase that exhibit various differences in their electron acceptor 



Si ^ .005 



-.05 .;C - 



1.0 . 1.5 

 [Loctate] ;jM 



Fig. 1. Relationship between the concentration of lactate and the rate of 

 appearance of the absorbance band of reduced cytochrome b.^ at A'^C and 25"C. 

 Double-beam spectrophotometer (424-404 m/x) ; 0-15 m phosphate buffer (pH 

 8-5). At 4^C, the rate of absorbancy change of cytochrome b^ proceeds at 

 0-16 /iM Fe/sec; at 25°, the rate is 008 /tM Fe/sec. Lactate concentrations appear 

 on the scale of the abscissa (calculated as 0-5 DL-lactate concentration). Rates 

 calculated for Ae 424 - 404 - 150cm-i x nrnr^ (Expt. 540). 



specificity, little experimental evidence is at hand to indicate the extent to which the 

 haemoprotein portion of this enzyme participates in the electron transfer process. A 

 single experiment is reported here in which the maximum speed with which lactate 

 alone can reduce the haemoprotein component is recorded. 



The results, illustrated by Fig. 1, show the relationship between the lactate con- 

 centration and the rate of increase of absorbancy measured at 424 m/< with reference 

 to 404 m/t. The absorbancy changes are computed as /<moles of iron/I./sec with 

 the approximate extinction coefficient (for this preparation, a value of approximately 

 150 cm-i X mM-i is used for 424 and 404 m/t)- The important feature of the experi- 

 ment is that the rate of reduction of the haemoprotein becomes independent of the 

 lactate concentration at concentrations above 0-5 /<m at the lower temperature, and 

 above 2-5 /tM lactate at the higher temperature. If the rate of reduction of the haemo- 

 protein is divided by its concentration to get an effective value of turnover number, 

 values of 004 and about 1 sec-^ are obtained at 4°C and 25°C respectively. Since it is 

 clear that this value is so inferior to the value observed in ferricyanide reduction (about 

 220 sec-^ at 26°C) and the comparable value in cytochrome c reduction, we suggest 

 that direct intramolecular electron transfer from flavin to haem is too slow to function 

 in a pathway for the overall enzymic activity. 



It may then be questioned whether there is intramolecular electron transfer at all, 

 and suggested that the reduction of the haematin may be a purely intermolecular 

 reaction, and this point requires further experimentation. In the presence of electron 

 acceptors such as ferricyanide, the rate of reduction of the haematin is somewhat 

 more rapid and it is feasible that the intermolecular electron transfer is facilitated 

 under these conditions. 



' In summary, the flavohaemoprotein cytochrome b.^ shows only a slow rate of haema- 

 tin reduction on adding lactate in the absence of electron acceptor. The system lacks 

 the property of rapid intramolecular electron transfer characteristic of the intact 



