期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：14
页码：E1697-E1704
DOI：10.1073/pnas.1423098112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceSoluble guanylate cyclase is the mammalian endogenous nitric oxide (NO) receptor that controls numerous signaling physiological processes. Time-resolved spectroscopy allowed us to probe the dynamics of the heme coordination after NO release and binding. After photodissociation of NO, all heme transitions are identified within the time range of 1 ps to 0.2 s, notably the bond breaking and reformation between the heme iron and proximal His, which are major events for the activation/deactivation processes. It is thus possible to demonstrate that the structural allosteric transition occurs in the time range 1-50 s, which remarkably matches the time range observed for hemoglobin, the prototypic protein for allostery. These findings relate not only to NO signaling but also to general allostery in heme proteins. We investigated the changes of heme coordination in purified soluble guanylate cyclase (sGC) by time-resolved spectroscopy in a time range encompassing 11 orders of magnitude (from 1 ps to 0.2 s). After dissociation, NO either recombines geminately to the 4-coordinate (4c) heme ({tau}G1 = 7.5 ps; 97 {+/-} 1% of the population) or exits the heme pocket (3 {+/-} 1%). The proximal His rebinds to the 4c heme with a 70-ps time constant. Then, NO is distributed in two approximately equal populations (1.5%). One geminately rebinds to the 5c heme ({tau}G2 = 6.5 ns), whereas the other diffuses out to the solution, from where it rebinds bimolecularly ({tau} = 50 s with [NO] = 200 M) forming a 6c heme with a diffusion-limited rate constant of 2 x 108 M-1*s-1. In both cases, the rebinding of NO induces the cleavage of the Fe-His bond that can be observed as an individual reaction step. Saliently, the time constant of bond cleavage differs depending on whether NO binds geminately or from solution ({tau}5C1 = 0.66 s and {tau}5C2 = 10 ms, respectively). Because the same event occurs with rates separated by four orders of magnitude, this measurement implies that sGC is in different structural states in both cases, having different strain exerted on the Fe-His bond. We show here that this structural allosteric transition takes place in the range 1-50 s. In this context, the detection of NO binding to the proximal side of sGC heme is discussed.
