Storey B T
Plant Physiol. 1970 Apr;45(4):455-60. doi: 10.1104/pp.45.4.455.
The half-times of oxidation by oxygen pulses of reduced cytochromes a and a(3) in mung bean mitochondria made anaerobic with succinate have been measured by means of a rapid mixing flow apparatus coupled to a dual wave length spectrophotometer in the presence and absence of cyanide. The absorbance changes at 438 to 455 millimicrons and 603 to 620 millimicrons are suitable for recording the time course of cytochrome a oxidation; the half-time is 2.0 milliseconds at 24 Celsius. This half-time does not change over the range 0 to 300 mum KCN, but the fraction of cytochrome a oxidized falls to a limiting value of 0.3 at the higher cyanide concentrations. The absorbance changes at 445 to 455 millimicrons record the time course of both cytochrome a and cytochrome a(3) oxidation; the former contributes 60% of the absorbance change and the latter 40%. The half-time for a(3) oxidation is calculated as 0.9 milliseconds at 24 Celsius. This half-time increases slightly to 1.3 milliseconds at 300 mum KCN. Reduced cytochrome a(3), whether uncomplexed or complexed with cyanide, becomes fully oxidized. The dissociation constant for the reduced cytochrome a(3)-cyanide complex is estimated to be 30 mum, whereas that for the oxidized a(3)-cyanide complex which inhibits electron transport is estimated to be 2 mum. This suggests two different binding sites for cyanide on the reduced and oxidized forms of cytochrome a(3). The fact that a limiting fraction of reduced cytochrome a can be oxidized at high cyanide concentrations implies that there is no interference by cyanide with electron transport from a to a(3), if cyanide remains bound to the site it occupies on reduced a(3) after this carrier becomes oxidized on reaction with molecular oxygen. Rearrangement of cyanide from this noninhibitory site to the inhibitory site occurs rapidly enough to compete with cytochrome a oxidation. The half-time for the rearrangement is calculated to be 0.9 milliseconds.
利用与双波长分光光度计相连的快速混合流动装置，在有和没有氰化物存在的情况下，测定了用琥珀酸使绿豆线粒体厌氧时，还原型细胞色素a和a₃被氧脉冲氧化的半衰期。在438至455毫微米和603至620毫微米处的吸光度变化适合记录细胞色素a氧化的时间进程；在24摄氏度时半衰期为2.0毫秒。在0至300微摩尔氰化钾范围内，该半衰期不变，但在较高氰化物浓度下，被氧化的细胞色素a的比例降至极限值0.3。在445至455毫微米处的吸光度变化记录了细胞色素a和细胞色素a₃氧化的时间进程；前者贡献吸光度变化的60%，后者贡献40%。在24摄氏度时，a₃氧化的半衰期计算为0.9毫秒。在300微摩尔氰化钾时，该半衰期略有增加至1.3毫秒。还原型细胞色素a₃，无论是否与氰化物结合，都会完全被氧化。还原型细胞色素a₃ - 氰化物复合物的解离常数估计为30微摩尔，而抑制电子传递的氧化型a₃ - 氰化物复合物的解离常数估计为2微摩尔。这表明氰化物在细胞色素a₃的还原型和氧化型上有两个不同的结合位点。在高氰化物浓度下，还原型细胞色素a的有限部分可以被氧化，这一事实意味着如果氰化物在该载体与分子氧反应被氧化后仍与它在还原型a₃上占据的位点结合，那么氰化物不会干扰从a到a₃的电子传递。氰化物从这个非抑制性位点重排到抑制性位点的速度足够快，足以与细胞色素a氧化竞争。重排的半衰期计算为0.9毫秒。
