As we all known,the instantaneous reaction between protein and ligands are very important to adjust the normal playing of biological function.And nitric oxide interactions with iron are the most important biological reactions in which NO participates.Unlike carbon monoxide or oxygen,NO can also bind reversibly to ferric iron.In this paper,UV-Vis absorption and CD spectra were used to study coordination reaction process between horse heart metMb and NO,to demonstrate the coordination reaction mechanism and to explore the influencing factors of metMb with NO.The experimental results showed that metMb could react with NO,and obtained three new peaks at 420 nm,534 and 568 nm,respectively,which implied metMb and NO have reacted and generated a new complex-nitrosylmetmyoglobin(metMbNO).Then as time went on,NO concentration decreased in the solution,and the Fe-N bond fractured under the attack of H2O,then NO leaves slowly from metMbNO,and metMb was regenerated.In this experiment,we also found that external conditions such as buffer medium,ionic strength,pH,temperature,etc,had an important influence on the coordination reaction between metMb and NO.It was favorable for the coordination reaction,when the 0.01 mol·L-1 phosphate buffer solution is near neutral condition,the temperature is 280 K,the coordination reaction could reach equilibrium at a fastest speed.In addition,the CD date show that NO only reacts with Fe atom in the center of heme and has less effect on the secondary structuers of protein.The research of metMb and NO played an important role to further study the function of NO.Especially the establish of equilibrium reaction mechanism between NO and heme protein has an important research value on maintaining the balance of NO in vivo and keeping the normal function in the body’s cells.