Lopez, known as 'Superman', heads the Vuelta general classification by 14 seconds from Primoz Roglic and 23sec from former winner Nairo Quintana after finishing fourth on Wednesday.
The 25-year-old has pulled clear of a group of contenders for overall victory including Spanish world champion Alejandro Valverde (fourth overall) three kilometres from the finish in Javalambre.
"I had a good feeling and I wanted to capitalise on the right moment," said Colombian Lopez, who is also the leader in the young riders' classification.
"We are happy with the work we did today. We are feeling good and everything is going well."
The day's winner Madruzo, part of a three-pronged Spanish breakaway group, claimed the first ever Vuelta stage victory for Spanish team Burgos.
In doing so he also consolidated his grip on the climbers' standings.
"To win here is one of the biggest achievements as a cyclist and for me it’s become a dream come true," said Madrazo, who burst into tears as he crossed the line for a huge win, his first in a Grand Tour.
"We have suffered throughout the season. The team deserved it, this goes for the team."
The 31-year-old's win comes after he struggled to keep pace in the category one Javalambre climb before bursting through for a thrilling victory.
Thursday sees the riders take on the hilly 199-kilometre sixth stage between Mora de Rubielos and the picturesque Valencian village of Ares del Maestrat, nestled at an altitude of 1,195 metres above sea level.